1 


■  NRLF 


Studies  in  the  Nitrogen  Metabolism 
of  Bacteria 


A  THESIS 

Submitted  to  the  Department  of  Chemistry  and  the  Committee  on 

Graduate  Study  of  the  Leland  Stanford  Junior  University 

in  partial  fulfilment  of  the  requirements  for 

the  degree  of  Doctor  of  Philosophy 


By 
H.  J.  SEARS 


Stanford  University,  January,  1916 

,"'■'           OF  THE  \ 

•    ^      UNIVEkSJTY     ■ 
"OF 


CHICAGO 

American    Medical    Association  • 

Five    Hundred    and    Thirty-Five    North    Dearborn    Street 

1916 


Studies  in  the   Nitrogen  Metabolism 
of   Bacteria 


A  THESIS 

Submitted  to  the  Department  of  Chemistry  and  the  Committee  on 

Graduate  Study  of  the  Leland  Stanford  Junior  University 

in  partial  fulfilment  of  the  requirements  for 

the  degree  of  Doctor  of  Philosophy 


By 
H.  J.  SEARS 


Stanford  University,  January,  1916 


'^''^-^tH^^'UJp. 


STUDIES    IN    THE    NITROGEN    METABOLISM    OF 

BACTERIA* 

H.    J.    Sears 

From    the   Department    of  Bacteriology   and   Immunology    of  Stanford    University,    California 

A  chemical  study  of  the  metabolism  of  any  organism  is  generally 
understood  to  mean  a  study  of  the  food  materials,  the  changes  which 
take  place  in  these  materials  within  the  organism,  the  agencies  which 
bring  about  these  changes,  and  the  character  and  composition  of  the 
excretory  products.  It  is  obvious  that  the  food  materials  of  micro- 
organisms may  be  as  completely  studied  as  those  of  higher  forms.  To 
determine  the  changes  these  undergo  within  the  cell,  however,  and  the 
exact  composition  of  the  excretory  products  is  a  problem  of  a  much 
more  difficult  nature  than  the  same  task  would  be  in  the  case  of  the 
higher  plant  and  animal  species. 

At  present,  students  of  bacterial  metabolism  must  content  them- 
selves solely  with  the  study  of  the  beginning  and  the  end  of  the  process. 
What  takes  place  within  the  cell  can  only  be  surmised  from  the  nature 
of  the  enzymes  which  have  been  expressed  from  the  bacterial  bodies 
and  from  the  composition  of  the  products  of  bacterial  action.  Fur- 
thermore, it  must  be  recognized  that  the  substances  found  in  a  bac- 
terial culture  medium  after  a  period  -of  growth  need  not  all  neces- 
sarily represent  the  end  products  of  metabolism.  There  are  numerous 
possibilities  for  alteration  of  the  true  metabolic  products  by  means  of 
their  mutual  action  upon  one  another. 

In  the  knowledge  of  these  difficulties,  therefore,  we  shall  have  to 
interpret  the  title  of  this  paper  to  mean  merely  a  study  of  the  nitrog- 
enous constituents  of  the  food  supply  of  bacteria  and  a  chemical 
examination  of  the  products  of  the  action  of  bacteria  upon  these  food 
substances. 

There  has  been  no  attempt  on  the  part  of  investigators  to  make  a 
complete  study  of  the  metabolism  of  any  micro-organism.  In  fact, 
such  a  study  would,  in  most  cases,  include  so  wide  a  variety  of  food 
materials  and  metabolic  end  products  that  the  large  amount  of  work 
involved  would  hardly  be  in  keeping  with  the  benefit  to  be  gained  from 


365230 


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4  H.    J.     Sears 

it.  Knowledge  of  the  subject  has  been  obtained,  therefore,  largely 
by  indirect  routes,  through  researches  undertaken  with  some  other 
object  in  view. 

The  phenomenon  of  putrefaction  has  long  been  a  subject  for  chemical 
research,  the  impetus  to  its  investigation  being  derived  partly  from  its  relation 
to  processes  taking  place  in  the  intestinal  tract  of  man  and  animals  and  partly 
from  its  relation  to  the  preservation  of  food-stuffs.  Earlier  experiments  were 
all  of  a  general  nature,  however,  being  carried  out  either  on  spontaneously 
putrefying  masses  or  on  pure  proteins  inoculated  from  such  masses.  The  only 
results  were  the  recognition  of  a  number  of  compounds  as  characteristic 
putrefactive  products,  and  the  isolation  of  many  toxic  substances.  Much  benefit 
was  derived  from  these  investigations  by  the  subjects  of  medicine  and  bio- 
logical chemistry,  but  little  was  added  by  them  to  the  knowledge  of  bacterial 
metabolism.  More  productive  in  this  direction  has  been  some  of  the  work  of 
later  years,  in  much  of  which  both  pure  proteins  and  pure  cultures  of  bacteria 
have  been  used. 

The  search  for  new  and  better  culture  media,  or  for  media  adapted  to  the 
growth  of  certain  species  of  micro-organisms,  has  been  responsible  for  many 
valuable  contributions,  particularly  to  the  knowledge  of  what  sort  of  nitrog- 
enous compounds  can  be  utilized  by  bacteria.  Likewise,  attempts  to  differenti- 
ate species  by  taking  advantage  of  the  dissimilarities  in  their  nitrogen  require- 
ments and  by  noting  the  different  products  resulting  from  the  decomposition 
of  the  same  nitrogenous  substance  by  different  species,  have  led  to  a  more 
careful  investigation  of  nitrogen  sources  and  of  the  end  products  of  nitrogen 
metabolism.  It  is  to  these  attempts  that  we  owe  the  extensive  data  to  be 
obtained  on  the  subject  of  indol-formation  by  bacteria,  as  well  as  on  their 
reducing  and  fermenting  powers.  In  recent  years  the  subject  of  creatinin- 
formation  has  been  studied  with  the  same  object  in  view. 

It  is  not  likely  that  it  is  possible  for  any  organism  to  grow  and  reproduce 
without  any  source  of  nitrogen  in  its  food  supply,  tho  Fermi*  asserted  that  he 
had  cultivated  a  micro-organism  containing  no  nitrogen  in  its  body  substance. 
In  the  form,  however,  in  which  this  nitrogen  may  be  offered  there  is  extremely 
wide  variation.  As  has  been  known  since  the  work  of  Berthelot^  in  1899,  there 
are  many  species  which  thrive  with  no  other  source  of  nitrogen  than  the  uncom- 
bined  atmospheric  nitrogen.  On  the  other  hand,  there  are  species  which  will 
accept  such  compounds  as  the  chitin*  of  plant  and  animal  origin,  as  their  only 
source  of  this  element. 

And  not  only  do  we  notice  this  variation  in  the  sources  of  nitrogen  among 
a  large  number  of  species,  but  even  with  one  and  the  same  species  it  is  now 
well  known  that  compounds  of  very  different  degrees  of  complexity  may  be 
utilized.  The  same  organism  may  grow  with  a  native  protein,  a  peptone, 
amino-acids,  amides  such  as  urea,  or  even  with  ammonium  salts,  as  its  only 
source  of  nitrogen.  Even  B.  tuberculosis,  an  organism  formerly  supposed  to 
be  exacting  in  its  cultural  requirements,  has  recently  been  grown  successfully 
on  a  medium  containing  nitrogen  only  in  the  form  of  ammonium  compounds.* 

If  bacteria  show  great  variations  in  their  choice  of  food  materials,  so  also 
do  they  show  wide  differences  in  the  ways  in  which  they  alter  these  materials 

»  Schmidt  and  Weis:  Die  Bacterien,   1902,  p.   102. 

*  Chimie  vegetale  et  agricole,   1899,   1. 

8  Benecke:  Botan.  Ztg.,   1905,  63,  p.  227. 

♦  Wherry:  Jour.  Infect.  Dis.,  1913,   13,  p.  144.     Kendall,  Day,  and  Walker:  Ibid.,  1914, 
15,  p.  417. 


Nitrogen     Metabolism     of    Bacteria  5 

in  the  course  of  metabolism,  and  in  the  kinds  of  chemical  products  which  they 
yield  by  means  of  such  alteration.  Two  factors  must  always  be  considered  in 
studying  the  chemical  products  of  bacterial  action ;  namely,  the  species  of  the 
organism  and  the  nature  of  the  substance  being  acted  upon.  Upon  the  same 
substrate  different  species  may  yield  very  different  products ;  likewise,  as  would 
be  expected  from  a  unicellular  organism,  the  same  species  may  yield  quite  dif- 
ferent products  when  grown  on  media  of  different  chemical  compositions.  The 
actual  chemical  processes  involved  in  the  decomposition  of  nitrogenous  com- 
pounds by  bacteria  are  difficult  to  study.  Equations  which  have  been  written 
to  represent  such  decompositions  must,  for  the  most  part,  be  placed  in  the  class 
of  speculations.  Such  speculations  are  of  great  value,  however,  and,  no  doubt, 
frequently  arrive  very  close  to  the  truth. 

That  proteolysis  through  the  agency  of  bacteria  capable  of  attacking  native 
proteins  pursues  the  same  general  course  as  that  brought  about  by  the  digestive 
enzymes  of  the  alimentary  tract  of  animals  seems  to  have  been  established 
beyond  dispute.  Emmerling  and  Rieser°  showed  that  B.  fluorescens-liquefaciens 
digested  gelatin  with  the  formation  of  proteoses  and  peptones.  These  were 
later  broken  down  to  lower  compounds  yielding  in  the  course  of  a  month  25% 
of  their  nitrogen  in  the  form  of  ammonia.  Substitution  compounds  of  ammonia 
were  also  found  in  the  form  of  methylamin,  trimethylamin,  betain,  and  cholin. 
That  amino-acids  were  an  intermediary  product,  however,  was  evidenced  by 
the  fact  that  they  were  able  to  identify  arginin  and  leucin.  Cultures  of  the 
same  organism  on  fibrin  solutions  contained  tyrosin,  leucin,  arginin,  and 
aspartic  acid.  Emmerling"  identified  the  amino-acids,  tyrosin  and  leucin,  in 
cultures  of  virulent  streptococci  on  blood  fibrin.  Mono-  and  trimethylamins 
were  present  here  also,  as  well  as  pyridin  bases.  According  to  Taylor,'  B.  coli 
digests  pure  casein  mainly  to  proteoses  and  peptones,  no  appreciable  quantities 
of  animo-acid  being  formed.  On  the  egg-meat  mixture  employed  by  Rettger' 
this  organism  produced  profound  changes,  giving  rise  to  the  aromatic  com- 
pounds indol  and  skatol,  the  amino-acids  tyrosin,  leucin,  and  tryptophan  being 
identified  as  intermediary  products.    Proteoses  and  peptones  were  formed  also. 

In  the  decomposition  of  proteins  the  obligate  anaerobes  play  a  most  impor- 
tant part.  In  fact,  according  to  Rettger,"  true  putrefactive  changes  with  the 
production  of  the  foul-smelling  mercaptans  and  hydrogen  sulfid  are  brought 
about  only  by  this  class  of  organisms,  the  part  played  by  the  aerobes  and 
facultative  anaerobes  being  that  of  creating  an  oxygen-free  environment  and 
removing  the  waste  products  of  the  strict  anaerobes.  From  his  researches  it 
appears  that  B.  putrificus,  B.  oedematis,  and  the  bacillus  of  symptomatic  anthrax 
are  the  most  powerful  putrefying  organisms  among  the  commoner  anaerobes. 
B.  tetanus  and  B.  welchii  have  little  or  no  putrefactive  power,  the  latter  being 
primarily  a  fermenting  organism. 

The  decomposition  of  the  primary  products  of  protein  hydrolysis  by  bacteria 
has  been  studied  but  little,  altho  mixtures  of  peptones  and  proteoses  sold  as 
peptone  have  long  been  the  favorite  basic  substance  in  bacterial  culture  media. 
By  means  of  the  change  in  the  rotation  of  polarized  light,  Abderhalden. 
Pincussohn,  and  Walther"  studied  a  number  of  the  common  pathogens   with 

■■■  Ber.  d.  deutsch.  chem.   Gesellsch.,   1902,  35,  p.  702. 
"  Ibid.,   1897,  30,  p.   1863. 
'  Ztschr.  f.  physiol.  Chem.,   1902,  36,  p.  487. 
s  Am.  Jour.  Physiol.,  1903,  8,  p.  284. 

»  Rettger  and  Newall:  Jour.  Biol.  Chem.,  1912,  13,  p.  341.     Rettger:  Ibid.,  1906,  2,  p.  71; 
1908,  4,  p.  45. 

'0  Ztschr.  f.  physiol.  Chem.,  1910,  68,  p.  471. 


6  H.     J.     Sears 

respect  to  the  extent  to  which  they  break  down  peptones  prepared  from  pure 
proteins,  and  compared  the  results  with  the  effect  on  the  proteins  themselves. 
Kendall  and  his  co-workers"""  recently  studied  the  production  of  ammonia  by  a 
large  number  of  species,  using  in  some  cases  Witte's  peptone  and  in  others  a  pep- 
tone solution  containing  meat  juice.  Their  aim  was  mainly  to  investigate  the  effect 
that  carbohydrates  have  on  the  decomposition  of  the  nitrogenous  substances. 
They  took  the  ammonia-production  as  a  measure  of  proteolysis.  Their  data 
show  interesting  exceptions  to  the  general  rule  that  carbohydrates  have  a 
protein-sparing  effect. 

The  investigation  by  Glenn^'  of  the  inhibition  of  indol-formation  by  members 
of  the  proteus  group  grown  in  a  peptone-carbohydrate  solution  also  indicates 
that  these  compounds  materially  lessen  proteolysis.  The  author  attributes  this 
effect,  however,  to  the  inactivation  of  the  tryptic  enzymes  of  the  bacteria  by  the 
acid  products  of  sugar-fermentation.  The  decreased  gelatin-liquefaction  by  this 
group  in  the  presence  of  sugars  fermentable  by  them  he  explains  in  the 
same  way. 

Berghaus'"  also  published  extensive  data  on  the  subject  of  ammonia-formation 
by  bacteria.  He  furthermore  drew  curves  representing  the  production  of  this 
compound  after  chemical  inhibition  of  growth. 

Kendall  and  Farmer"  attempted  also  to  measure  the  rate  of  the  production 
of  amino-acid,  but  were  unable  with  the  method  used  (formol  titration)  to 
get  results  of  any  value. 

Kendall  and  Walker"  claimed  the  production  of  minute  quantities  of  urea 
from  meat-juice  peptone  solutions,  and  further  stated  that  the  amounts  formed 
day  by  day  were  about  proportional  to  the  ammonia  produced. 

Antonoff,**  using  Weyl's  test,  and  German,"  using  Salkowski's  method,  claimed 
creatinin-production  from  Witte's  peptone  for  a  large  number  of  species.  Both 
investigators  believed  the  tests  to  have  differentiating  value.  Fitzgerald  and 
Schmidt^  repeated  these  tests,  but  could  find  appreciable  amounts  of  creatinin 
only  in  cultures  of  B.  proteus.  They  employed  both  Weyl's  method  and 
Jaffe's  picric-acid  test. 

That  polypeptids  are  produced  by  bacteria  has  not  been  established  as  far 
as  I  know.  That  they  may  be  utilized,  however,  as  a  source  of  nitrogen  is 
known.  Sasaki^  demonstrated  the  ability  of  a  variety  of  species  to  split  some 
of  the  simpler  peptids  into  their  constituent  amino-acids. 

A  study  of  the  metabolism  of  bacteria  grown  on  media  containing  nitrogen 
only  in  the  form  of  amino-acids  has  been  productive  of  much  information  that 
is  interesting  and  valuable.  We  may  deal  here  with  synthetic  as  well  as  analytic 
products.     That  proteins  are  synthesized  from  amino-acids  by  micro-organisms 

"  Kendall  and  Farmer:  Jour.   Biol.  Chem.,  1912,  12,  pp.   13,   19,  21,  465,  469. 
»*  Kendall,  Farmer,  Bagg,  and  Day:  Ibid.,  p.  219. 
i»  Kendall  and  Farmer:  Ibid.,  1912,  13,  p.  64. 

'«  Kendall,  Day,  and  Walker:  Jour.  Infect.  Dis.,  1913,  13,  p.  425. 
•»  Kendall  and  Walker:  Jour.  Biol.  Chem.,  1913,  IS,  p.  277. 
'"  Kendall,  Day,  and  Walker:  Jour.  Med.  Research,  1913,  28,  p.  465. 

«'  Kendall.  Day,  and  Walker:  Jour.  Am.  Chem.  Soc,  1913,  35,  pp.  1201,  1208,  1211, 
1217,  1225,  1237. 

'»  Centralbl.  f.  Bakteriol.,  I,  O.,   1911,  58,  p.  481. 

'»  Arch.  f.  Hyg.,  1908,  64,  p.   1. 

•-»  Centralbl.  f.  Bakteriol.,  I,  O.,  1906,  43,  p.  209. 

=>  Ibid.,  1912,  63,  p.  545. 

=2  Proc.  Soc.  Exper.   Biol,  and  Med.,  1912,  10,  p.   55. 

-'  Biochem.   Ztschr.,  1912,  41,  p.    174;    1913,  47,  pp.   462.   472. 


Nitrogen     Metabolism     of    Bacteria  7 

follows  as  a  matter  of  course  when  we  say  that  growth  is  supported  by  them. 
That  proteins  other  than  those  of  the  bacterial  bodies  are  formed  seems  to  be 
true  also."*  Moreover,  it  has  been  shown  by  Frankel'"  and  others'"  that  the 
characteristic  toxins  of  diphtheria  and  tetanus  are  formed  in  media  containing 
only  amino-acids  as  a  source  of  nitrogen. 

The  fraction  of  the  nitrogen  of  the  amino-acid  that  is  used  in  synthesis  is 
always  very  small.  The  greater  portion  is  found  in  the  form  of  simpler  com- 
pounds. Frouin  and  Ledebt"  grew  several  species  on  the  amino-acids  resulting 
from  the  hydrolysis  of  serum  proteins  and  observed  that  in  all  cases  a  primary 
acidity  was  produced  which  was  followed  later  by  strong  alkalinity.  Rivas*" 
found  that  a  short  digestion  of  peptone  with  trypsin  made  it  a  much  better 
culture  medium  than  the  undigested  peptone.  On  such  a  medium  he  obtained 
indol  reactions  in  from  5  to  6  hours. 

Of  the  amino-acids  which  have  been  used  alone  as  a  source  of  nitrogen 
for  micro-organisms,  asparagin  has  been  most  studied.  A  very  large  number 
of  organisms  are  capable  of  utilizing  this  compound.  The  main  manner  of 
decomposition  is  deaminization  with  formation  of  aspartic  acid  and  a  subsequent 
production  of  ammonia  from  the  latter.  That  nitrogenous  products  other  than 
ammonia  are  usually  formed  also  is  probable.  Nawiasky'"*  made  a  rather 
exhaustive  study  of  the  action  of  B.  proteus  on  asparagin  when  large  quantities 
of  the  organisms  are  added  to  pure  asparagin  solutions.  The  most  of  the 
nitrogen  was  recovered  in  the  form  of  ammonia.  About  5%  of  the  asparagin 
which  disappeared  was  not  accounted  for  by  the  ammonia  recovered. 

Tyrosin  is  broken  down  by  B.  coli  and  yields  78.7%  of  the  theoretical  amount 
of  p-oxyphenylethylamin.'"  Traetla  Mosca"  found  another  organism  which 
decomposed  this  acid  with  the  formation  of  p-hydrocoumaric  acid  and  ammonia. 

Of  the  nitrogenous  compounds  other  than  amino-acids,  special  interest 
attaches  to  those  found  in  more  or  less  abundance  in  the  urine  of  man  and 
animals.  That  urea,  uric  acid,  and  hippuric  acid  are  attacked  by  a  number  of 
species  of  bacteria  has  long  been  known.  Kossiwicz""  showed  that  a  number 
of  molds  were  capable  of  utilizing  these  substances  also.  Liebert"  found  sev- 
eral varieties  of  bacteria  that  decompose  uric  acid  to  ammonia,  and  he  stated 
that  allantoin  and  urea  were  intermediary  products.  Certain  other  organisms 
have  been  isolated"  which  yield  only  urea  from  uric  acid,  no  ammonia  being 
formed. 

That  a  very  large  number  of  species  exist  capable  of  converting  urea  to 
ammonium  carbonate  is  evident  from  the  researches  of  Miquel."  It  is  prob- 
able also  that  many  of  the  common  laboratory  forms  show  this  property.' 

"  Muller:  Pfliiger's  Arch.,  1906,  112,  p.  245. 

»  Hyg.  Rundschau,  1894,  4,  p.  769. 

■•'•  Uschinsky:  Centralbl.   f.  Bakteriol.,  1893,  14,  p.  316.     Arch,  de  nied.  exper.  et  d'anat. 

path.,  1893,  5,  p.  293. 

"  Compt.  rend.  Soc.  de  biol.,  1911,  70,  p.  24. 

»  Centralbl.  f.  Bakteriol.,  I,  O.,  1912,  63,  p.  547. 

-•  Arch.  f.  Hyg.,  1908,  66,  p.  209. 

•0  Sasaki:   Biochem.  Ztschr.,   1914,  59,  p.  429. 

«  Gazz.  chim.  Ital.,  1910,  40,  p.  86. 

"  Ztschr.  Gahrungsphysiol.,  I,  60,  and  II,  51. 

»  Botan.  Centralbl.,  1910,  114,  p.  361. 

"  Ulpiani:  Jahrb.  f.  Tierchem.,  1903,  33,  p.  1034.  Gerard:  Compt.  rend.,  1896,  122, 
p.   1019;   123,  p.   185. 

''  Lafar's  Handbuch  der  technischen  Mykologie,  1904-1906,  3,  p.   71. 


8  H.     J.     Sears 

Creatinin  is  attacked  slowly  by  bacteria'"  as  is  creatin  also.  Nawiasky  showed 
that  the  latter  was  decomposed  by  B.  proteus  only  to  the  extent  of  8.64%. 
Only  3.69%  of  the  amount  attacked  was  accounted  for  by  the  ammonia  pro- 
duced.    He  assumed  that  methylguanidin  was  formed. 

THE     PRODUCTION     BY     BACTERIA    OF    AMINO-ACID     AND     AMMONIA 

FROM     PEPTONE 

That  ammonia  is  the  chief  end  product  of  the  nitrogen  metaboHsm 
of  bacteria  seems  to  have  been  well  established.  That  the  ammonia- 
production  by  an  organism  growing  on  a  protein  or  peptone  medium 
is  always  a  measure  of  the  organism's  proteolytic  activity  cannot,  from 
this  fact,  be  assumed  to  be  true.  It  is  quite  conceivable  that,  because 
of  the  differences  in  the  rate  of  the  decomposition  of  the  primary 
products  of  proteolysis,  this  criterion  might  lead  us  astray.  We  might, 
for  instance,  have  an  accumulation  of  amino-acids  in  the  medium  and 
a  very  slight  production  of  ammonia,  or,  on  the  other  hand,  a  decom- 
position of  the  amino-acids  as  fast  as  formed  with  a  consequently 
high  concentration  of  ammonia.  It  would  give  a  better  idea,  therefore, 
of  the  rate  and  extent  of  protein-decomposition  if  data  were  secured 
on  the  concentrations  of  both  amino-acid  and  ammonia.  The  new 
method  originated  by  Van  Slyke^^  for  determining  amino-acid  nitrogen 
now  makes  the  procuring  of  such  data  possible.  The  analytical 
results  of  the  examination  of  a  large  number  of  cultures  with  respect 
to  their  change  day  by  day  in  amino-acid  and  ammonia  content  are 
given  in  the  following  pages  by  means  of  tables.  Some  are  shown  also 
in  the  form  of  curves. 

The  free  ammonia  was  determined  by  Folin's  aeration  method,  in  which 
Ca(0H)2  is  used  to  set  the  ammonia  free  from  its  salts.  After  the  ammonia 
had  been  completely  removed,  the  sample  was  filtered  off  from  the  excess 
calcium  hydroxid  and  a  determination  of  the  amino-acid  was  made  by  Van 
Slyke's  micro  method.  The  Kjeldahl-Gunning- Arnold  method  was  used  for 
total-nitrogen  determinations. 

The  first  organisms  investigated  were  the  strongly  putrefactive  facultative 
anaerobes,  B.  proteus-vulgaris  and  B.  pyocyaneus.  The  medium  used  was  a 
solution  containing  2%  peptone  and  0.5%  NaCl.  Five  hundred  cubic  centi- 
meters of  this  solution  were  placed  in  each  of  two  1000-c.c.  flasks.  After  ster- 
ilization in  the  autoclave  at  15  pounds'  extra  pressure,  they  were  inoculated  and 
placed  in  an  incubator  at  Z7  C.  By  means  of  a  sterile  pipet  a  sample  was  with- 
drawn from  each  immediately  after  inoculation,  and  at  intervals  of  24  hours 
thereafter  for  11  days.  These  samples  were  analysed  at  once  for  free  ammonia 
and   amino-acids.     Creatinin   was   also   determined   in   the    samples    from   the 

'«  Ackermann:  Ztschr.   Biol.,  1913,  62,  p.  208;  63,  p.  78. 
"  Jour.   Biol.  Chem.,  1913,  16,  p.  161. 


Nitrogen     Metabolism     of    Bacteria  9 

culture  of  B.  proteus.  Tests  for  this  compound  in  the  cultures  of  B.  pyocyaneus 
were  all  negative.  Folin's"  method  was  employed  for  the  determination  of 
creatinin. 

Table  1  gives  the  analytical  data  for  this  test.  The  figures  represent 
the  total  amount  in  milligrams  of  the  substance  mentioned  at  the  head 
of  the  column,  that  is  present  in  100  c.c.  of  the  culture  fluid  on  the 
corresponding  day.  The  third  column  under  each  organism  gives  the 
ratio  between  the  amounts  of  amino-acid  nitrogen  and  ammonia 
nitrogen  present  on  each  day  of  the  test.  Chart  1  represents  the  same 
results  in  the  form  of  curves. 

The  data  show  very  marked  differences  between  the  two  organ- 
isms in  their  action  on  peptone  solutions.  In  the  culture  of  B.  proteus 
we  notice  for  the  first  2  days  a  decrease  in  the  amino-acid  already 
present  in  the  medium,  followed  by  a  rise  in  concentration  on  the 
3rd,  4th  and  5th  days.  Thereafter  the  concentration  rises  and  falls 
without  any  tendency  to  get  very  far  from  a  mean  value  of  about  50 
mgm.  per  100  c.c.  The  ammonia  nitrogen  also  decreases  for  the  first  2 
days,  but  thereafter  rises  rapidly  until  the  end  of  the  experiment,  reach- 
ing the  concentration  of  nearly  70  mgm.  per  100  c.c.  The  ratio  between 
the  two  forms  of  nitrogen  decreases  rapidly  throughout  the  test. 

The  culture  of  B.  pyocyaneus  likewise  shows  an  initial  decrease  in 
its  amino-acid  nitrogen,  followed  by  fluctuations  up  and  down  until 
the  6th  day,  after  which  there  occurs  a  gradual  rise  in  concentration 
until  the  end  of  the  experiment  on  the  10th  day.  The  free  ammonia 
also  suffers  a  decrease  in  this  culture  in  the  first  24  hours.  Thereafter 
there  is  a  general  tendency  toward  a  slow  increase  of  this  substance, 
but  in  two  instances,  on  the  4th  and  7th  days,  a  decrease  occurs.  The 
ratio,  amino-acid  nitrogen  to  ammonia  nitrogen,  falls  in  this  case  also, 
but  the  decline  is  much  slower  than  in  the  case  of  B.  proteus,  and  the 
ratio  does  not,  in  the  time  of  the  experiment,  reach  nearly  so  low  a 
value.  These  differences  are  made  plainer  by  the  curves.  The  amino- 
acid  curve  of  B.  pyocyaneus  tends  to  rise  rapidly.  The  free  ammonia 
curve  of  B.  proteus  rises  very  rapidly;  that  of  B.  pyocyaneus  is  much 
more  gradual. 

It  is  interesting  to  note  that  both  cultures  show  an  initial  decrease 
in  both  their  amino-acid  nitrogen  and  their  ammonia  nitrogen,  indicat- 
ing that,  for  purposes  of  growth  and  reproduction,  these  organisms 
select  the  simpler  forms  of  nitrogen  in  preference  to  the  more  complex 
peptone  and  proteose  molecules.    This  seems  to  be  in  accord  with  the 

"»  Jour.  Biol.  Chetn.,  1914,  17,  p.  469. 


10 


H.    J.     Sears 


.    0 

i 

g  70 

1-4 

"   69 


B.  proteus -vulgaris 


/ 


/ 


r 


«i     ^ — ^       / 


B.pyocijaoQJS 


3     ^      <x      9     7      e 

AK  or  CULTUHI  III  DtY8 


/©      /o 


Chart   1.     Production  of  amino-acid  and  ammonia  by   bacteria  in  a  2%   peptone   solution 
=  ammonia  nitrogen; =   amino-acid  nitrogen. 


Nitrogen     Metabolism     of    Bacteria 


11 


finding  of  Sperry  and  Rettger*^  that  pure  proteins  are  not  attacked  by 
micro-organisms  in  the  entire  absence  of  simpler  nitrogenous  com- 
pounds. 

Simultaneously  with  the  foregoing  experiment  another  was  made 
using  the  same  two  organisms  and  also  B.  coli-communis,  but  employ- 
ing a  peptone  solution  containing  meat  juice  instead  of  the  pure  peptone 
media.  The  medium  contained  in  1  liter  the  juice  from  1  lb.  of  finely 
ground  lean  beef,  10  gm.  of  Witte's  peptone,  and  5  gm.  of  sodium 
chlorid.  The  reaction  was  made  neutral  to  phenolphthalein.  The 
technic  of  the  experiment  was  exactly  similar  to  that  described.  Table 
2  and  Chart  2  give  the  analytical  results  of  the  test.  As  in  Table  1 
the  values  are  given  in  milligrams  per  100  c.c.  of  the  culture  fluid. 

TABLE     1 
The   Production   of  Amino-acid  and   Ammonia  by   Bacteria   in  a  2%   Peptone   Solution 


B.  Proteus-Vulgaris 

E 

.  Pyocyaneus 

Age  of 

Ratio: 

Ratio: 

Culture 

Amlno- 

Am- 

Amino- 

Amino- 

Am- 

Amino- 

in  Days 

acid 

monia 

acid  N 

Creat- 

acid 

monia 

acid  N 

Nitro- 
gen 

Nitro- 
gen 

inin 

Nitro- 
gen 

Nitro- 
gen 

Am- 

Am- 

monia N 

monia  N 

0 

42.0 

13.3 

4.65 



42.0 

13.3 

1 

34.0 

7.3 

4.52 

— 

39.9 

5.7 

7.00 

2 

32.9 

7.3 

4.52 

— 

48.2 

7.3 

6.62 

3 

41.8 

13.0 

3.22 

— 

49.2 

9.2 

5.35 

4 

45.3 

15.6 

2.91 

— 

42.7 

7.3 

5.86 

5 

56.5 

18.2 

3.10 

— 

36.6 

12.2 

3.00 

6 

51.2 

22.7 

2.26 

Trace 

50.5 

20.0 

2.53 

7 

60.8 

34.6 

1.76 

Trace 

56.1 

19.0 

2.95 

8 

57.9 

41.4 

1.40 

Trace 

68.8 

19.5 

3.54 

9 

61.2 

45.3 

1.13 

Trace 

80.6 

24.7 

3.26 

10 

48.9 

59.6 

0.82 

Trace 

83.7 

26.0 

3.22 

11 

46.2 

69.2 

0.67 

Trace 

Ttie  figures  represent  the  amount  in  milligrams  of  the  substance  mentioned  at  the  bead 
of  the  column,  that  is  present  in  100  c.c.  on  the  corresponding  day. 


The  chief  differences  between  the  data  of  Table  2  and  those  of 
Table  1  are  to  be  seen  in  the  case  of  the  cultures  of  B.  pyocyaneus. 
In  the  presence  of  the  muscle  extractives  this  organism  shows  a  con- 
siderably higher  production  of  ammonia  and  a  much  lower  production 
of  amino-acid.  There  is  no  tendency  at  all  toward  an  accumulation  of 
the  latter  in  the  medium.  Evidently  in  the  case  of  this  bacillus  the 
constituents  of  the  meat  juice  have  a  marked  protein-sparing  effect. 
The  rapidly  decreasing  figures  for  creatinin  indicate  that  this  compound, 
at  least,  is  easily  utilized. 

'»  Jour.  Biol.  Chem.,  1915,  20,  p.  445. 


12 


H.     J.     Sears 


20 

to 

o 


Sugar  ^r9€Z*^  Pt^tann  Solution  Conitxin/n^  r^ertErrtrvC.-^ 
CrrnQtinina.  Oocomposi'h'on 


d>/>yoc/0/w  US 


B.  coli'CO'f'nijnts 


-^mrrtonfaf\fitroyn 

AOC  OF  CULTURE  IN  BAYS 

Chart  2.     The  key  to  this  chart  applies  also  to  Charts  3,  4,  and  5. 


Nitrogen     Metabolism     of    Bacteria 


13 


B.  proteus  gives  very  similar  curves  for  ammonia  and  amino-acid, 
as  it  did  in  the  2%  peptone  solution,  the  ammonia  values  being  high 
and  the  amino-acid  values  comparatively  low,  but  showing,  however, 
a  tendency  toward  a  maximum  about  the  6th  day.  On  account  of 
the  ability  of  this  organism  to  produce  creatinin  from  peptone,  the 
values  for  this  compound  cannot  be  taken  to  indicate  the  extent  to 
which  the  extractives  are  utilized. 

B.  coli  shows  somewhat  high  ammonia  values ;  in  view  of  the  com- 
parative volumes  of  the  cultures,  they  are  enormously  higher  than  the 
values  on  corresponding  days  shown  by  this  organism  in  pure  peptone 
solutions  (see  Table  4). 

TABLE     2 

The  Production  of  Amino-acid  and  Ammonia  by   Bacteria   in   Meat-Extract 
Peptone  Solutions 


Age 

of 
Cul- 

B. Proteus 

B. 

Pyocyaneus 

B.  Coli 

Amino- 

Am- 

Amino- 

Am- 

Amino- 

Am- 

ture 

acld 

monia 

Creat- 

acid 

monia 

Creat- 

acid 

monia 

Creat- 

in 

Nitro- 

Nitro- 

inin 

Nitro- 

Nitro- 

inin 

Nitro- 

Nitro- 

inin 

Days 

gen 

gen    . 

gen 

gen 

gen 

gen 

0 

35.0 

14.9 

53.1 

35.0 

14.9 

53.1 

35.0 

14.9 

53.1 

1 

36.1 

18.7 

51.2 

41.8 

17.9 

50.7 

33.6 

13.5 

52.7 

2 

29.1 

16.2 

51.0 

21.0 

6.8 

48.0 

29.9 

15.6 

51.2 

3 

42.4 

21.1 

48.5 

31.4 

16.2 

47.8 

46.7 

18.4 

50.6 

4 

41.6 

24.7 

45.8 

25.3 

16.2 

42.7 

36.0 

19.3 

46.5 

5 

55.9 

47.4 

39.4 

35.2 

28.4 

33.1 

38.5 

20.6 

40.1 

6 

65.1 

56.0 

33.8 

38.4 

33.9 

16.2 

35.3 

24.2 

35.0 

7 

61.7 

60.2 

30.3 

55.1 

39.18 

13.6 

39.5 

28.4 

29.8 

8 

42.8 

60.2 

27.6 

56.6 

50.8 

11.7 

50.6 

29.6 

26.1 

9 

38.5 

65.7 

27.6 

37.6 

56.5 

9.2 

54.1 

32.5 

25.2 

10 

30.1 

65.0 

26.1 

27.2 

53.6 

7.0 

51.2 

35.4 

18.6 

11 

23.1 

31.0 

24.4 

52.1 

6.0 

50.9 

42.2 

16.1 

As  none  of  the  organisms  used  in  the  described  tests  reached  the 
limits  of  its  chemical  activity  in  the  time  of  the  experiment,  it  was 
thought  worth  while  to  repeat  the  experiment,  continuing  it  over  a 
longer  period.  Furthermore,  as  it  was  thought  possible  that  the 
removal  of  samples  with  a  pipet  every  24  hours  might  introduce  errors 
by  disturbing  the  culture  or  through  failure  to  take  account  of  slight 
differences  that  might  exist  in  the  concentration  of  the  substances 
determined  in  different  layers  of  the  solution,  the  following  rather 
tedious  method  was  used: 

A  2%  peptone  solution  containing  0.5%  NaCl  was  made  up  and  placed  in 
40-c.c.  portions  in  small  Erlenmeyer  flasks.  These,  after  sterilization  in  the 
autoclave,  were  all  placed  in  the  incubator  at  Zl  C.  Each  day  2  flasks  were 
inoculated  with  1  loopful  of  a  24-hour  peptone  culture  of  B.  proteus  and  B. 
pyocyaneus,  respectively.  In  this  way  separate  cultures  were  obtained  ranging 
in  age  from  1  to  28  days,  all  of  which  had  been  inoculated  with  approximately 


14 


H.    J.     Sears 


the  same  number  of  organisms  and  kept  for  the  full  time  of  the  experiment 
under  exactly  the  same  conditions.  When  all  but  the  control  fiask  had  been 
inoculated,  they  were  sterilized  by  the  addition  of  about  2%  phenol.  Two 
cubic  centimeters  of  N/1  HCl  were  added  to  each  and  the  cultures  filtered.  The 
filtrate,  which  was  fairly  clear,  was  made  up  to  volume  and  aliquot  parts  taken 
for  the  determinations.  Table  3  gives  the  analytical  results.  As  in  Tables  1 
and  2,  the  values  are  given  in  milligrams  per  100  c.c.  of  the  culture  medium. 

TABLE     3 
The  Production  or  Amino-acid  and  Ammonia  by  Bacteria  in  a  Peptone  Solution 


B.  Proteus-Vulgaris 

B.  Pyocyaneus 

Age  nt 

Ratio: 

1 

Ratio: 

Culture 

Amino- 

Am- 

Amino- 

Amino- 

Am- 

Amino- 

in 

acid 

monia 

acid  N 

Great-         Crea- 

acid 

monia 

acid  N 

Days 

Nitro- 
gen 

Nitro- 
gen 

inln              tin 

Nitro- 
gen 

Nitro- 
gen 

Am- 

Am- 

monia N 

monia  N 

0 

37.0 

6.5 

5.70 

37.0 

6.5 

5.70 

1 

62.0 

24.2 

2.56 

44.4 

14.5 

3.06 

2 

60.0 

25.0 

2.40 

Trace 

89.4 

21.8 

4.10 

8 

54.5 

40.5 

1.84 

Trace             6.8 

112.0 

28.1 

5.14 

4 

60.0 

40.5 

1.48 

Trace           10.5 

63.1 

25.4 

2.48 

5 

56.5 

58.7 

1.05 

Trace 

84.3 

?8.1 

3.00 

(> 

59.7 

59.0 

1.01 

Trace 

80.0 

31.8 

2.52 

7 

43.2 

81.2 

0.54 

Trace 

116.0 

37.2 

8.12 

S 

52.5 

96.2 

0.55 

Trace     i       14.0 

59.8 

39.8 

1.50 

» 

39.5 

92.5 

0.48 

6.1       i       .... 

120.0 

51.0 

2.36 

10 

39.5 

81.5 

0.49 

6.0 

59.2 

46.7 

1.27 

11 

32.5 

104.0 

0.82 

6.5 

101.0 

65.7 

1.54 

12 

87.0 

108.0 

0.85 

6.5                16.5 

53.0 

43.8 

1.21 

13 

120.0 

.... 

6.5          : 

70.5 

47.9 

1.47 

14 

27.5 

122.0 

6.'23 

7.3        

81.8 

69.7 

1.17 

15 

27.0 

124.0 

0.22 

6.3        i 

98.0 

70.0 

1.33 

16 

81.4 

122.0 

0.26 

6.0                14.4 

189.0 

71.7 

1.94 

17 

30.0 

106.0 

0.28 

7.7        1 

97.6 

49.0 

1.99 

18 

27.0 

127.0 

0.21 

7.3 

102.0 

63.5 

1.61 

19 

20.0 

114.0 

0.19 

7.3 

100.0 

68.5 

1.58 

20 

28.8 

137.0 

0.21 

8.0 

107.0 

60.0 

1.78 

21 

29.8 

86.7 

0.35 

104.0 

59.0 

1.76 

22 

25.0 

111.0 

0.23 

sis               i4!2 

49.0 

50.7 

0.97 

23 

23.7 

159.0 

0.15 

.    4.8 

45.4 

89.0 

1.15 

24 

22.5 

136.0 

0.16 

6.7                 9.5 

60.0 

54.5 

1.10 

25 

27.0 

112.0 

0.24 

8.2 

60.0 

38.1 

1.58 

26 

24.5 

109.0 

0.22 

7.0 

65.0 

27 

22.0 

97.4 

0.23 

7.5        

59.7 

28 

22.5 

Sl.O 

0.28 

6.0                  4.5 

■ 

64!6 

49.8 

i.28 

A  number  of  interesting  facts  are  brought  out  by  this  experiment.  In  the 
first  place,  the  ammonia  figures  do  not,  in  either  case,  indicate  the  relative  ages 
of  the  cultures.  The  10-day  culture  of  B.  proteus,  for  example,  shows  a  lower 
concentration  of  ammonia  nitrogen  than  the  8-day  culture.  Likewise,  the 
12-day  culture  of  B.  pyocyaneus  contains  a  lower  concentration  of  this  compound 
than  the  9-day  culture.  Many  other  instances  of  the  same  irregularity  may 
be  noted.  It  is  apparent,  therefore,  that  different  cultures  of  the  same  organ- 
ism on  the  same  media  and  in  exactly  equal  volumes  may  show  quite  different 
rates  of  ammonia-formation,  even  when  made  and  grown  under  exactly  the 
same  conditions.  In  the  case  of  both  organisms,  however,  there  is  a  general 
increase'  of  ammonia-formation  on  the  part  of  the  older  cultures  up  to  the 
15th  or  16th  day.  After  this  the  values  fluctuate  without  any  regular  increase. 
Evidently,  in  the  volunxe  of  media  used,  these  organisms  reach  their  maximum 
of  ammonia-production  in  from  about  14  to  18  days. 


Nitrogen     Metabolism     of    Bacteria 


15 


The  amino-acid  figures  are  comparable  with  those  of  Table  1.  B.  proteus 
gives  a  fluctuating,  but  consistently  low  value,  while  B.  pyocyaneus  gives  a 
relatively  high  value,  which  shows  a  tendency  to  increase  with  the  age  of  the 
culture.  The  ratios,  when  the  volumes  concerned  are  taken  into  account,  show 
practically  the  same  characteristics  as  those  in  Table  1. 

An  experiment  similar  to  the  foregoing  was  carried  out  also  on  the  three 
organisms,  B.  coli-communis,  B.  typhosus,  and  Sp.  cholerae.  The  technic  was 
identical  with  that  described  in  the  case  of  B.  proteus  and  B.  pyocyaneus. 
Table  4  gives  the  data  on  this  experiment. 

TABLE     4 
The  Production  of  Amino-acid  and  Ammonia  by  Bacteria  in  a  Peptone  Solution 


Age 

of 
Cul- 
tures 

in 
Days 

B.  Coli-Communls 

B.  Typhosus 

Sp.  Cholerae 

Amino- 
acid 

Nitro- 
gen 

Am- 
monia 
Nitro- 
gen 

Ratio: 
Amino- 
acid  N 

Amino- 
acid 

Nitro- 
gen 

Ratio: 
Am-        Amino- 
monia      acid  N 
Nitro 

Amino- 
acid 

Nitro- 
gen 

Am- 
monia 
Nitro- 
gen 

Ratio: 
Amino- 
acid  N 

Am- 
monia N 

gen           Am- 
monia N 

Am- 
monia N 

0 

2 

3 

4 

5 

6 

8 

10 

12 

15 

18 

21 

30.8 
27.7 
28.6 
26.8 
35.7 
34.4 
90.6 
37.1 
31.1 

47!8 
45.0 

7.1 
11.6 
7.7 
5.4 
9.3 
9.7 
10.9 
13.7 
15.4 
16.6 
16.9 
24.2 

4.34 
2.39 
3.74 
5.01 
3.84 
3.55 
2.90 
2.71 
2.02 

2.'83 
1.86 

30.8 

39.3 

36.2 

35.6, 

32.2 

30.4 

31.2 

34.8 

36.6 

31.7 

31.2 

33.9 

7.1           4.23 
12.8            3.07 
9.3            3.89 

9.8  3.64 

12.8  2.52 

10.9  2.79 

9.9  3.16 
11.6      1      3.00 

9.3            3.94 
11.6            2.74 
21.0            1.49 
13.9            2.44 

30.8 
37.9 
45.7 
51.9 
65.6 
73.2 
35.7 
97.7 

m'.i 

7.1 
12.5 
15.7 
22.0 
29.2 
29.2 
21.0 
34.7 
49.9 
52.2 
52.0 
46.0 

4.34 
3.03 
2.91 
2.36 
2.25 
2.51 
1.70 
2.82 

2,'26 

Little  comment  is  necessary  on  these  figures.  The  irregularity  in  the  rate 
of  ammonia-production  which  was  mentioned  in  connection  with  Table  3  is 
found  here  also.  It  will  be  observed,  however,  that  the  figures  representing 
the  concentrations  of  amino-acid  nitrogen  deviate  very  little  in  the  case  of 
B.  coli  and  B.  typhosus  from  those  found  with  uninoculated  medium.  In  the 
culture  of  Sp.  cholerae  there  is  a  decided  increase  of  amino-acid  nitrogen  with 
the  age  of  the  culture.  As  would  be  expected,  the  ammonia-production  by  this 
organism  is  relatively  high  also. 

With  the  same  technic  as  that  just  described,  cultures  were  examined  of 
B.  proteus-vulgaris  on  a  1%  peptone  solution  containing  10  gm.  of  Liebig's  meat 
extract  per  liter.  In  addition  to  amino-acid  nitrogen  and  ammonia,  both  creatin 
and  creatinin  were  determined  in  this  case.  Table  5,  which  gives  the  analytical 
data  for  the  test,  shows  that  on  this  medium  also  there  is  a  very  great  irregu- 
larity in  the  production  of  ammonia.  It  is  impossible  to  estimate  from  these 
data  at  what  age  cessation  of  chemical  activity  occurs.  The  creatinin  values 
would  indicate  that  this  compound  is  continuously  decomposed  throughout  the 
entire  period  of  37  days.  The  fluctuating  values  for  creatin  are  undoubtedly 
due,  in  part,  to  the  different  degrees  to  which  this  substance  is  decomposed  in 
sterilization. 

It  is  evident  from  these  examples  that  this  method  of  procedure 
is  unsuited  to  the  quantitative  study  of  the  pcptolytic  activity  of  micro- 
organisms.    Succeeding  experiments  were  carried  on,  therefore,  with 


16 


H.     J.     Sears 


the  technic  employed  in  the  original  experiments ;  namely,  that  of  inocu- 
lating a  large  volume  of  the  medium  and  withdrawing  samples  day  by 
day  with  a  sterile  pipet.  It  was  further  determined  also  to  study  the 
effect  of  glucose  on  amino-acid-production.  It  had  been  shown  clearly 
by  Kendall  and  his  co-workers  that  this  carbohydrate  very  considerably 
reduced  the  rate  of  ammonia- formation  by  most  micro-organisms.  It 
was  a  matter  of  interest  to  know  also  whether  it  would  reduce  amino- 
acid-production. 

TABLE     5 

The  Production  ok  Ami.noacid  and  Ammonia  by  B.  Proteus-Vulgabis  in  a  Meat-Extract 

Peptone   Solution 


Age  of 

Amlno- 

Ratio: 

Amino-acid  N 

Culture 

acid 
Nitrogen 

Ammonia 
Nitrogen 

Amino-acid  N 

Creatinin 

Creatln 

plus 
Ammonia  N 

'n 

Days 

Ammonia  N 

0 

38.8 

11.6 

3.34 

62.0 

48.0 

50.4 

2 

65.8 

15.0 

4.35 

61.0 

43.0 

80.8 

4 

70.5 

36.2 

1.95 

57.2 

39.8 

106.7 

5 

69.5 

46.4 

1.50 

57.0 

43.0 

115.9 

8 

38.8 

50.8 

0.76 

37.6 

47.9 

89.6 

» 

30.6 

58.0 

0.53 

34.1 

54.6 

88.6 

11 

42.5 

48.0 

0.88 

29.4 

56.1 

90.5 

13 

86.5 

55.2 

0.66 

30.3 

44.7 

91.7 

14 

36.5 

52.0 

0.70 

39.0 

41.0 

88.5 

15 

29.8 

51.7 

0.58 

38.1 

53.9 

81.5 

18 

28.4 

26.9 

1.05 

27.6 

54.9 

55.3 

21 

41.8 

67.7 

0.62    ■ 

30.8 

50.7 

109.5 

23 

34.6 

45.2 

0.76 

27.8 

46.2 

79.8 

26 

19.1 

28.2 

0.68 

23.0 

53.5 

47.3 

29 

28.6 

36.2 

0.79 

19.6 

43.3 

64.8 

35 

30.1 

40.0 

0.75 

20.5 

47.1 

70.1 

37 

47.1 

17.9 

51.6 

Seven  organisms  were  investigated  in  this  respect.  The  exact  technic  of 
the  experiment  was  as  follows.  A  large  amount  of  a  2%  peptone  solution  con- 
taining 0.5%  NaCl  was  prepared  and  divided  into  2  equal  portions,  to  one  of 
which  was  added  approximately  1%  of  pure  glucose.  The  media  were  then 
placed  in  500-c.c.  flasks,  300  c.c.  to  each.  To  each  of  the  flasks  containing 
glucose  a  small  amount  of  CaCos  was  added.  All  the  samples  were  sterilized 
together  in  the  autoclave  for  10  minutes  under  an  extra  pressure  of  15  lb. 
They  were  all  inoculated  at  Zl  C.  in  the  same  incubator.  The  incubator  was 
kept  saturated  with  moisture  to  prevent  evaporation.  Samples,  15  c.c.  in  amount, 
were  withdrawn  at  intervals  as  indicated  in  the  tables,  and  were  subjected  at 
once  to  a  half  hour's  heating  in  steam  at  100  C.  This  would,  of  course,  not 
sterilize  the  cultures  of  B.  subtilis,  but  as  the  analyses  were  usually  completed 
on  the  same  day  that  the  samples  were  taken  (otherwise  the  samples  were  kept 
in  the  ice  chest),  it  is  not  probable  that  any  error  was  introduced  by  that  fact. 

Tables  6  to  12  inclusive  give  the  results  of  the  analyses  of  these 
samples.  Charts  3  to  5  represent  the  same  results  in  the  form  of 
curves. 


Nitrogen     Metabolism     of    Bacteria  17 

An  examination  of  the  tables,  or  a  glance  at  the  curves,  shows  at 
once  that,  as  would  be  expected,  the  peptolytic  activity  of  B.  subtilis  is 
much  greater  than  that  of  any  of  the  rest,  Sp.  metchnikovii  being  the 
only  one  in  this  group  which  shows  a  comparable  production  of  amino- 
acid  or  of  ammonia.  It  is  unfortunate  that,  as  the  result  of  a  con- 
tamination on  the  7th  day,  the  examination  of  the  only  other  organism 
having  a  gelatin-liquefying  power,  namely.  Staphylococcus  pyogenes, 
could  not  be  carried  out  over  the  full  period.  The  data  obtained,  how- 
ever, indicate  that  in  its  chemical  activity  it  is  to  be  compared  with  the 
nonliquefying  member  of  the  group  rather  than  with  those  mentioned. 

The  effect  of  glucose  on  the  nitrogen  metabolism  of  the  cultures  is 
apparent  in  both  the  amino-acid  and  the  free-ammonia  curves.  In  all 
cases,  except  those  of  B.  faecalis-alkaligenes  and  B.  dysenteriae,  there 
is  an  unquestionably  lower  ammonia-production  in  the  cultures  con- 
taining glucose.  The  cultures  of  B.  dysenteriae,  when  the  slightly  dif- 
ferent conditions  which  may  exist  in  the  two  flasks  are  taken  into 
account,  may  be  said  to  give  practically  identical  ammonia  curves.  The 
same  is  probably  true  of  B.  faecalis-alkaligenes,  altho  it  is  interesting  to 
note  that,  in  this  case,  the  curve  of  the  glucose-containing  culture  is 
consistently  above  that  of  the  one  containing  no  glucose. 

If  it  is  assumed  that  neither  of  the  organisms  just  mentioned  is 
capable  of  utilizing  glucose,  it  becomes  difficult  to  explain  the  lower 
concentrations  of  amino-acids  in  the  cultures  containing  this  carbo- 
hydrate. There  seem  to  be  but  two  possible  explanations  of  this  fact. 
Either  the  glucose  shows  a  protein-sparing  action  in  that  it  decreases 
the  peptone-decomposition  by  the  organisms,  or  it  acts  in  a  manner  that 
must  be  considered  the  direct  opposite  of  this.  That  is,  it  brings  about 
a  more  rapid  breaking-down  into  simpler  compounds  of  nitrogen  of  the 
amino-acids  resulting  from  the  splitting  of  the  peptone.  In  this  case 
it  is  necessary  to  assume  that  the  breaking  down  of  the  amino-acids 
is  not  carried  on  to  the  free-ammonia  stage  or  else  that  this  stage  is 
passed  and  free  nitrogen  or  the  oxids  of  nitrogen  are  formed. 

The  latter  theory  is  so  entirely  in  disagreement  with  all  the  well- 
known  biologic  reactions  of  glucose  that  it  may  safely  be  put  out  of 
consideration  at  once.  If  we  accept  then  as  an  explanation  of  the  facts 
the  theory  of  decreased  nitrogen  metabolism,  we  have  two  instances 
of  this  phenomenon  which  are  not  indicated  b)'  the  production  of  free 
ammonia. 


18 


H.    J.     Sears 


TABLE     6 
The  Formation  of  Amino-acid  and  Ammonia  fkom   Peptone  by  B.  Fakatyfhosus 

Total  Nitrogen   =r   304  mgm.  per  100  c.c. 


Without  Glucose 

With  Glucose 

Age  of 

Culture 

Amino- 

Ratio: 

Amino- 

Ratio: 

in 

acid 

Ammonia 

Amino-acid  N 

acid 

Ammonia 

Amino-acid  N 

Days 

Nitro- 
gen 

Nitro- 
gen 

Nitro- 
gen 

Nitro- 
gen 

Ammonia  N 

Ammonia  N 

0 

42.4 

3.5 

12.10 

42.4 

3.5 

12.10 

1 

56.7 

4.3 

13.20 

34.1 

2.0 

17.10 

2 

42.5 

4.4 

9.67 

38.6 

... 

3 

42.0 

3.2 

13.10 

28.5 

1.4 

20.20 

4 

35.7 

4.7 

7.61 

26.2 

4.3 

6.06 

5 

7.8 

34.0 

3.0 

11.30 

6 

36.3 

29.6 

4.5 

6.56 

7 

32.0 

8.i 

3.95 

81^ 

6.2 

5.08 

9 

31.9 

9.2 

3.47 

28.2 

11 

33.1 

9.0 

3.67 

35.4 

2.4 

14.80 

14 

41.0 

9.9 

4.14 

34.2 

4.1 

8.35 

18 

42.7 

9.1 

4.68 

39.2 

2.0 

•19.70 

TABLE    7 

The  Formation  of  Amino-acid  and  Ammonia  from   Peptone  by   B.   Acidi-lactici 

Total  Nitrogen   =i   304  mom.  per  100  c.c. 


Age  of 

Culture 

in 

Days 

Without  Glucose 

With  Glucose 

Amino- 
acid 

Nitro- 
gen 

Ammonia 
Nitro- 
gen 

Ratio: 
Amino-acid  N 

Amino- 
acid 

Nitro- 
gen 

Ammonia 
Nitro- 
gen 

Ratio: 
Amino-acid  N 

Ammonia  N 

Ammonia  N 

0 

1 

2 

3 

4 

5 

6 

7 

9 

11 

14 

18 

42.4 
42.2 
49.5 
47.9 
88.0 
87.6 
29.9 
41.6 
84.5 
41.7 
47.8 
53.5 

3.5 

0.0 

2.8 

4.4 

4.7 

8.1 

6.8 

9.7 

12.2 

12.8 

14.4 

18.4 

12.10 

ii'.io 

10.90 
8.08 
4.63 
4.40 
4.28 
2.83 
8.89 
8.82 
2.91 

42.4 
38.3 
44.5 
30.6 
28.8 
32.1 
34.6 
28.2 
36.8 
29.8 
33.9 

3.5 
0.0 
3.0 
1.7 
5.9 
8.0 
6.4 
0.0 

aie 

4.8 
5.2 

12.10 

i2!86 
26.20 
5.19 
9.60 
5.96 

ioio 

6.22 
6.52 

TABLE     8 
The   Formation   of   Amino-acid  and  Ammonia  from   Peptone   by   B. 
Total  Nitrogen   =  304  mgm.  per  100  c.c. 


F^calis-Alkaligenes 


Age  of 

Culture 

in 

Days 

Without  Glucose 

With  Glucose 

Amino- 
acid 

Nitro- 
gen 

Ammonia 
Nitro- 
gen 

Ratio: 
Amino-acid  N 

Amino- 
acid 
Nitro- 
.  gen 

Ammonia 
Nitro- 
gen 

Ratio: 
Amino-acid  N 

Ammonia  N 

Ammonia  N 

0 

1 

2 

3 

4 

5 

6 

7 

9 

11 

14 

18 

42.4 
37.7 
52.8 
34.9 
25.0 
30.0 
31.5 
35.8 
36.8 
38.8 
38.9 
88.0 

3.5 
0.0 
2.6 
4.6 
5.1 
5.9 
7.0 
6.4 
6.1 
5.3 
3.4 
5.2 

12.10 

26!36 
7.59 
4.90 
5.08 
4.50 
5.59 
6.04 
7.23 

11.40 
6.36 

42.4 
28.6 
36.1 
33.8 
32.6 
25.0 
25.2 
19.0 
27.5 
23.0 
28.0 
26.5 

3.5 
3.5 
3.5 
4.9 
5.7 
5.8 
7.7 
12.2 
6.5 
5.1 
5.1 
4.9 

12.10 
8.17 

10.30 
6.91 
5.72 
4.32 
4.42 
1.56 
4.23 
4.51 
5.49 
5.42 

TO 

to 

30 

S    20 


A 


BL  pa  rat  typhosus 


8 


S 


60 


I    SO 

30 

ZO 

fO 

O 


/---. 


v\ 


-N 


/' 


B. acidi  lactic i 


«v 

r 

< 
\ 

/ 

y,.* 

-4. 

90 

'^x' 

K 

'^v 

jf^ 

-x-^ 

V-^Vi^-. 

-«v. 

^A 


S    ^ 


4> 

(0 


B.faecaiis  alKallgene% 


/"' 


J0 


Budijsenterlae,  Shiga 


Chart   3.     The   formation   by  bacteria  of  amino-acid  and   ammonia   from   peptone. 


20 


H.     J.     Sears 


TABLE     9 

The   Formation   of  Amino-acid   and  Ammonia   from    Peptone  by   B.   Dysenteryi^e,    Shiga 

Total  Nitrogen   =   304  mom.  per  100  c.c. 


Age  of 

Culture 

in 

Dhys 

Without  Glucose 

With  Glucose 

Amino- 
acid 

Nitro- 
gen 

Ammonia 
Nitro- 
gen 

Ratio: 
Amino-acid  N 

Amino- 
acid 

Nitro- 
gen 

Ammonia 
Nitro- 
gen 

Ratio: 
Amino-acid  N 

Ammonia  N 

Ammonia  N 

0 

1 

2 

3 

4 

5 

6 

7 

9 

11 

14 

18 

42.4 
38.3 
49.1 
47.9 
36.6 
38.7 
26.5 
42.9 
44.3 
43.6 
37.9 
43.4 

3.5 
3.2 
3.8 
3.8 
4.7 
6.2 
4.2 
5.1 
7.9 
6.5 
9.7 
5.9 

12.1 
12.0 
12.9 
12.6 
7.8 
6.2 
6.3 
8.4 
5.6 
6.7 
3.9 
7.4 

42.4 
30.6 
34.9 
32.8 
30.2 
32.9 
30.8 
31.0 
25.8 
33.2 
27.4 
27.5 

3.5 
2.6 
1.5 
7.6 

h'.i 

6.6 
5.4 
8.8 
5.6 
6.3 
2.6 

12.1 

14.2 

33.0 

4.3 

5.4 
4.7 
5.7 
2.9' 
6.0 
4.4 
10.6 

TABLE     10 

The  Formation  of  Amino-acid  and  Ammonia  from  Peptone  by  Staphylococcus  Pyogenes 
Total  Nitrogen   =   304  mgm.  per  100  c.c. 


Age  of 

Culture 

in 

Days 

Without  Glucose 

With  Glucose 

Amino- 
acid 

Nitro- 
gen 

Ammonia 
Nitro- 
gen 

Ratio: 
Amino-acid  N 

Amino- 
acid 

Nitro- 
gen 

Ammonia 
Nitro- 
gen 

Ratio: 
Amino-acid  N 

Ammonia  N 

Ammonia  N 

0 
1 

2 
3 
4 
5 
6 
7 
8 
11 
14 
18 

42.4 
43.4 
43.2 
50.7 
39.3 
46.2 
31.8 
42.0 

8.5 
3.6 
5.2 
4.9 
8.5 
7.3 

i'.i 
... 

12.1 
12.0 
8.3 
10.3 
4.6 
6.3 

'5.5 

42.4 
37.5 
42.9 
43.5 
35.9 
40.6 
45.5 
41.0 
47.3 
47.8 
56.2 
62.5 

3.6 
3.2 
1.2 
3.5 
4.1 
2.8 
4.5 
10.2 
6.1 
6.0 
5.5 
5.2 

12.1 
11.7 
36.6 
12.4 

8.8 
14.5 
10.1 
40.2       • 

7.8 

8.0 
10.2 
12.0 

TABLE     11 

The  For.mation  of  Amino-acid  and  Ammonia  from  Peptone  by  Sp.  Metchnikovii 

Total  Nitrogen   =:   304  mgm.  per  100  c.c. 


Age  of 

Culture 

in 

Days 

Without  Glucose 

With  Glucose 

Amino- 
acid 

Nitro- 
gen 

Ammonia 
Nitro- 
gen 

Ratio: 
Amino-acid  N 

Amino- 
acid 

Nitro- 
gen 

Ammonia 
Nitro- 
gen 

Ratio: 
Amino-acid  N 

Ammonia  N 

Ammonia  N 

0 

1 

2 

8 

4 

6 

6 

7 

9 

11 

14 

18 

42.4 
37.3 
50.9 
51.4 
56.9 
49.5 
55.5 
42.0 
75.9 
92.4 
91.7 
113.2 

3.5 
2.2 
6.5 
11.9 
16.3 
21.7 
29.8 
L5.2 
27.0 
50.5 
41.6 
46.6 

12.1 
16.9 
7.8 
4.3 
3.7 
2.8 
1.8 
1.7 
2.7 
1.8 
2.2 
2.4 

42.4 
40.3 
39.1 
43.4 
41.2 
40.0 
43.9 
62.2 
40.0 
48.5 
52.8 
67.8 

3.6 

0.0 

1.2 

10.1 

9.3 

11.5 

11.1 

10.8 

11.7 

9.6 

11.3 

14.5 

12.1 

32.6 
4.3 
4.4 
3.5 
4.0 
5.8 
3.4 
6.1 
4.7 
4.7 

Nitrogen     Metabolism     of     Bacteria 


21 


Staph  tjlococc  us 
aureus 


7       8      9       10       u       il      13 

AOS    OF  OULTUKB    IH  DAYS 


iS     16     ti     la 


ii» 


/0» 


Spirillum  metchnlKovU 


5     ^      7     e     9     to    ff     /2    '^     '^    '^     ^^    '"^    '^ 

AOt  Of  CULTURI   IK  DAY3 
Chart  4.     The  formation  by  bacteria  of  amino-acid  and  ammonia  from  peptone. 


22 


H.     J.     Sears 


4  \ 


TABLE     12 

The  Formation  of  Amino-acid  ./^ij^^mmoma  from  Peptone  j|y  B.  Subtilis 
Total  Nitrogen-—   304  mgm.  per  100  c.c.  r 


Without  Glucose 

With  Glucose 

Age  of 

Culture 

Amino- 

Eatio: 

Amino- 

RaJio: 

In 

acid 

Ammonia 

Amino-acid  N 

acid 

Ammonia 

Amino-acid  \ 

Days 

Nitro- 
gen 

Nitro- 
gen 

Nitro- 
gen 

Nitro- 
gen 

Ammonia  N 

Ammonia  N 

0 

42.4 

3.5 

12.1 

42.4 

3.5 

12.1 

1 

44.1 

2.4 

18.4 

29.2 

2.0 

14.6 

2 

60.1 

13.9 

4.8 

12.1 

4.3 

9.8 

S 

79.4 

19.4          ;              4.1 

51.3 

4.4 

11.7 

4 

86.8 

29.4                        2.9 

50.2 

8.5 

5.9 

6 

94.0 

35.9          '              2.6 

65.8 

15.4 

4.3 

6 

100.0 

48.1 

2.0 

71.0 

20.1 

3.5 

7 

122.0 

58.1 

2.3 

92.3 

22.7 

4.1 

0 

117.5 

55.5 

2.1 

97.8 

31.1 

3.1 

11 

114.0 

62.5 

1.8 

129.3 

35.0 

3.7 

14 

101.2 

68.7 

1.5 

112.0 

37.8 

3.0 

18 

109.0 

65.9 

1.7 

135.8 

51.2 

2.6 

Chart  5.     The  formation  by  hacteri.n  of  amino-acid  and   ammonia   from   peptone. 


Nitrogen     Metabolism     of    Bacteria  23 

That  the  presence  of  glucose  might  decrease  the  peptone-splitting 
power  of  the  cultures  by  preventing,  to  some  extent,  the  reproduction 
of  the  organisms  is  contrary  to  experience,  and  would  furthermore 
make  the  identical  ammonia  curves  difficult  to  explain. 

The  decrease  during  the  first  24-48  hours  in  the  amount  of  free  ^ 
ammonia  which  was  originally  present  in  the  culture  medium  is  noticed 
in  the  case  of  all  the  organisms  studied.  This  decrease  seems  to  take 
place  to  practically  the  same  extent  in  the  cultures  containing  glucose. 
It  is  undoubtedly  true  that  in  such  cultures  the  most  rapid  multiplica- 
tion of  the  organisms  takes  place  during  the  first  24-48  hours.  It  is 
therefore  probable  that  this  ammonia  is  utilized  for  the  synthesis  of 
bacterial  protein.  That  ammonium  salts  are  so  utilized  by  certain 
micro-organisms  when  these  salts  are  their  only  source  of  nitrogen 
is  a  well-established  fact.  But  that  such  micro-organisms  as  those 
investigated  should  utilize  ammonia  in  the  presence  of  other  forms  of 
nitrogen  seems  at  first  a  little  surprising.  A  glance  at  the  curves  repre- 
senting the  concentrations  of  ammonia  in  the  glucose-containing  culture 
will  show,  however,  that  this  compound  is  continuously  utilized  by  the 
bacteria  in  question.  Frequent  decreases  in  concentration  take  place 
with  all  but  the  strongly  proteolytic  organism,  B.  subtilis.  That  the 
same  decreases  are  not  observed  in  the  cultures  not  containing  glucose 
is  probably  due  to  the  greater  proteolysis  in  the  latter  rather  than  to  an 
increased  assimilation  of  ammonia  in  the  presence  of  the  sugar.  It  is 
safe  to  assume  that  in  no  case  does  the  ammonia  found  represent  the 
entire  amount  formed  by  the  organisms  in  the  course  of  metabolism. 

Also,  an  initial  lowering  of  the  amino-acid  concentrations  is 
observed  in  most  of  the  cultures.  This  lowering  is  generally  greater  in 
the  glucose-containing  cultures,  a  fact  which  is  doubtless  explained  by 
the  decreased  proteolytic  activity  in  the  latter. 

In  the  cultures  of  B.  subtilis  and  Sp.  metchnikovii  there  is  a  ten- 
dency toward  accumulation  of  amino-acids,  both  in  the  presence  and  in 
the  absence  of  glucose.  In  the  case  of  B.  subtilis  this  accumulation 
takes  place  after  the  first  24  hours  at  about  an  equal  rate  in  both 
cultures  until  the  7th  day  is  reached.  After  this  the  culture  not  con- 
taining the  sugar  shows  a  decrease  while  the  one  containing  the  sugar 
continues  to  show  an  increase  at  about  the  same  rate.  The  maximal 
concentration  of  amino-acid  in  the  case  of  the  latter  is  not  passed  at 
the  end  of  the  experiment  on  the  18th  day. 

The  ammonia  curves  of  this  organism  are  roughly  parallel  to  the 
amino-acid  curves.    The  sum  of  the  amino-acid  nitrogen  and  ammonia 


24  H.'   J.     Sears 

nitrogen  in  the  non-glucose-containing  culture  on  the  7th  day  is  nearly 
the  same  as  the  sum  of  these  two  substances  in  the  glucose-containing 
culture  on  the  18th  day,  and  is  approximately  equal  to  3/5  of  the  total 
nitrogen  of  the  culture  medium.  Of  all  the  cultures  tested,  that  of 
Sp.  metchnikovii  shows  the  greatest  reduction  of  proteolytic  activity 
due  to  the  presence  of  glucose.  -  In  the  sugar-containing  culture  of  this 
organism  the  concentration  of  ammonia  is  uniformly  low  while  in  that 
free  of  sugar  it  reaches  a  value  of  50.5  mgm.,  or  about  17%  of  the 
total  nitrogen  of  the  medium.  The  amino-acid  content  of  this  culture 
attains  the  value  of  113.2  mgm.,  or  about  37%  of  the  total  nitrogen 
content.  Taking  into  account,  as  will  be  pointed  out  later,  the  presence 
of  other  simple  compounds  of  nitrogen,  we  can  say  that  this  organism 
in  the  absence  of  sugar  is  capable  of  decomposing  almost  completely 
a  2%  solution  of  peptone.  By  the  same  reasoning  B.  subtilis  may  be 
said  to  be  capable  of  this  even  in  the  presence  of  1%  of  glucose. 

THE  PRODUCTION  OF  AMINO-ACID  AND  AMMONIA  BY  BACTERIA  IN   MEDIA 

CONTAINING   GELATIN 

The  medium  used  in  these  tests  was  a  1%  peptone  solution  containing  0.5% 
of  NaCl  to  which  was  added  5%  of  pure  gelatin.  The  medium  was  cleared 
by  means  of  egg  white,  made  neutral  to  phenolphthalein,  and  filtered  until  clear. 
To  one  half  of  the  solution  so  prepared  1%  of  glucose  was  added.  Both  por- 
tions were  then  placed  in  300-c.c.  flasks,  200  c.c.  to  each.  Calcium  carbonate 
was  added  to  the  samples  containing  the  carbohydrate.  All  the  flasks  were 
sterilized  under  IS  pounds'  extra  pressure  for  10  minutes.  They  were  then  all 
inoculated  at  the  same  time  and  placed  together  in  the  same  incubator  at  27  C. 

All  the  organisms  used  were  capable  of  liquefying  gelatin  except  B.  faecalis- 
alkaligenes  and  B.  cloacae.  The  strict  anaerobe,  B.  aerogenes-capsulatus,  and 
the  facultative  anaerobe,  B.  pyocyaneus,  were  grown  under  anaerobic  conditions 
by  keeping  the  surface  of  the  medium  covered  with  a  thick  layer  of  sterilized 
paraffin  oil.  Samples  were  removed  at  definite  intervals  by  means  of  sterile 
pipets.  These  samples  were  heated  in  steam  at  100  C.  for  one-half  hour  and 
at  once  subjected  to  analysis. 

Tables  13  to  16  inclusive  give  the  analytical  results.  The  total 
nitrogen  of  the  sterile  medium  was  828.6  mgm.  per  100  c.c.  B.  subtilis 
shows  much  the  same  characteristics  in  its  action  on  gelatin  as  on  the 
peptone  solution.  It  produces  both  amino-acid  and  free  ammonia  much 
more  rapidly  at  first  in  the  absence  of  glucose,  but  the  rate  of  the 
production  of  these  substances  in  the  presence  of  the  sugar  increases 
in  the  later  stages  until,  at  the  end  of  the  5th  week,  the  concentration 
of  ammonia  nitrogen  is  actually  greater  in  this  culture  than  in  the 
culture  containing  no  glucose  and  the  concentration  of  the  amino-acid 
nitrogen  is  nearly  equal  to  that  in  the  culture  not  containing  glucose. 


Nitrogen     Metabolism     of     Bacteria 


TABLE     13 

The  Production  of  Amino-acid  and  Ammonia  from  Gelatin  by  Bacteria 

Total  Nitrogen  =  828.6  mgm.  per  100  c.c. 


Age 

in 

Days 

B.  Subtilis 

B.  Cloacae 

Without  Glucose 

With  Glucose 

Without  Glucose 

With  Glucose 

Amlno- 
acidN 

Am- 
monia N 

Amino- 
acldN 

Am-, 
monia  N 

Amino- 
acid  N 

Am- 
monia N 

Amino- 
acid  N 

Am- 
monia N 

0 

1     ■ 
•     2 

4 

7 
10 
15 
24 
34 

42.5 

79.2 
130.7 
206.8 
226.7 
225.1 
185.0 
172.0 

5.9 

iiU 

20.5 
65.2 
88.0 
73.5 
136.1 
183.4 

42.5 

65.6 
97.3 
120.4 
140.4 
189.0 
185.0 
159.5 

5.9 

'  "5.2 

5.9 

0.0 

14.9 

39.0 

113.0 

164.3 

42.5 
58.0 
45.8 
69.3 
71.8 
56.9 
63.7 
62.8 
65.5 

5.9 
0.0 
0.0 
5.4 
9.2 
22.2 
33.3 
36.7 
43.5 

42.5 
45.7 
38.2 
52.3 
63.3 
44.8 
50.8 
82.6 
56.5 

5.9 
1.3 
0.0 
0.0 
0.0 
9.1 
27.2 
24.0 
50.7 

TABLE     14 

The  Production  of  Amino-acid  and  Ammonia  from  Gelatin  by  Bacteria 

Total  Nitrogen  —  828.6  mgm.  per  100  c.c. 


B.  Faecalis-Alkaligenes 

B.  Pyocyaneus,  Aerobic 

Age 
in 

Without  Glucose 

With  Glucose 

Without  Glucose 

With  Glucose 

Days 

Amino- 

Am- 

Amino- 

Am- 

Amino- 

Am- 

Amino- 

Am- 

acid N 

monia  N 

acid  N 

monia  N 

acid  N 

monia  N 

acid  N 

monia  N 

0 

42.5 

5.9 

42.5 

5.9 

42.5 

5.9 

42.5 

5.9 

1 

51.8 

0.0 

1.8 

44.8 

0.0 

2 

60.5 

0.0 

85.5 

0.0 

50.2 

0.0 

44.8 

0.0 

4 

54.7 

0.0 

45.0 

0.0 

47.4 

2.8 

54.7 

0.0 

7 

36.4 

0.0 

38.8 

0.0 

87.6 

11.5 

48.7 

0.0 

10 

55.4 

2.3 

42.0 

2.8 

109.5 

21.9 

52.3 

0.0 

16 

39.9 

7.0 

3S.2 

6.5 

279.0 

34.2 

55.0 

1.0 

24 

63.7 

14.3 

42.6 

12.1 

145.0 

49.7 

113.0 

18.6 

34 

62.0 

14.7 

46.8 

18.1 

161.0 

41.2 

202.4 

35.5 

TABLE     IS 

The  Production  of  Amino-acid  and  Ammonia  from  Gelatin  by  Bacteria 
Total  Nitrogen  =:  828.6  mgm.  per  100  c.c. 


B 

Pyocyaneus 

,  Anaerobic 

B.  Welchii 

Age 

in 

Without  Glucose 

With  Glucose 

Without  Glucose 

With  Glucose 

Days 

Amino- 

Am- 

Amino- 

Am- 

Amino- 

Am- 

Amino- 

Am- 

acid N 

monia  N 

acid  N 

monia  N 

acid  N 

monia  N 

acid  N 

monia  N 

0 

42.5 

5.0 

42.5 

5.9 

42.5 

5.9 

42.5 

5.9 

1 

0.0 

0.0 

,     , 

2 

55.4 

48.6 

0.0 

47.1 

0.0 

49.3 

0.0 

4 

48.7 

0.0 

59.7 

0.0 

79.1 

0.0 

186.4 

19.9 

7 

56.0 

0.0 

53.1 

0.0 

36.4 

0.0 

260.7 

76.6 

10 

67.3 

5.7 

34.4 

2.8 

56.3 

0.0 

277.2 

92.2 

15 

94.0 

13.9 

89.0 

9.3 

61.3 

1.3 

305.0 

95.2 

24 

159.1 

34.7 

105.3 

17.8 

66.2 

3.2 

303.0 

85.7 

34 

194.2 

40.3 

187.9 

25.9 

53.7 

3.6 

292.0 

30.3 

26 


H.     J.     Sears 


TABLE     16 

The  Production  of  Amino-acid  and  Ammonia  from  Gelatin  by  Bacteria 
Total  Nitrogen  =  828.6  mgm.  per  100  c.c. 


Sp.  Cholerae 

Age  in  Days 

Without  Glucose 

With  Glucose 

Amino-acid 

N 

Ammonia 

N 

Amino-acid     i 

N 

Ammonia 

N 

0. ;- 

42.6 

48.0 

58.0 

48.7 

80.7 

123.8 

174.0 

237.0 

266.8 

6.9 

0.0 

3.9 

0.0 

1.76 

80.4 

36.8 

78.1 

100.7 

42.6 

49.5 
70.5 
40.7 
42.4 
41.8 
40.3 
45.1 

5,9 

1 

2 

0.0 

4 

0.0 

7 

0.0 

10 

0.2 

15 

4.2 

24 :.:: :: 

34 

8.3 
4.1 

The  initial  decrease  in  free  ammonia  is  not  observed  in  the  sugar- 
free  culture,  probably  because  of  the  fact  that  a  sample  was  not 
analyzed  at  the  end  of  one  day's  incubation.  This  decrease  is  apparent, 
however,  in  the  glucose-containing  culture,  and  the  latter  also  shows 
evidence  of  the  continuous  utilization  of  free  ammonia  in  the  falling 
off  of  the  concentration  from  5.9  mgm.  per  100  c.c.  on  the  4th  day  to  0 
on  the  7th.  Both  the  ammonia-  and  the  amino-acid-production  reach 
higher  values  than  in  the  corresponding  cultures  on  peptone  alone.  The 
sum  of  the  two  forms  of  nitrogen  in  the  sugar-free  culture  does  not 
differ  greatly,  on  the  last  day  of  the  experiment,  from  that  in  the  case 
of  the  sugar-containing  cultures,  the  two  sums  being  approximately 
2,7%  and  39%  respectively  of  the  total  nitrogen  of  the  sterile  medium. 

The  spirillum  of  Asiatic  cholera  offers  a  very  interesting  example 
of  the  protein-sparing  action  of  glucose.  In  the  concentrations  of 
both  amino-acid  and  free-ammonia  the  differences  beween  glucose 
and  nonglucose  cultures  are  enormous.  The  data  show  that  in  the 
absence  of  the  carbohydrates  the  proteolytic  activity  of  this  organism 
is  considerable,  as  great,  in  fact,  as  that  of  B.  subtilis  or  B.  pyocyaneus. 
With  glucose  present,  however,  the  indication  is  that  the  gelatin  is 
attacked  very  little,  if  at  all,  since  the  figures  are  much  lower  even 
than  those  shown  by  this  organism  on  a  pure  peptone  medium  (Table 
3).  It  is  evident  that  the  effect  of  the  sugar  is  continued  throughout 
the  entire  time  of  the  experiment.  That  chemical  activity  was  not 
brought  to  a  standstill  by  products  of  sugar-decomposition  is  evident 
from  the  fluctuating  values  of  ammonia  and  amino-acid  nitrogen. 


Nitrogen     Metabolism     of    Bacteria  27 

The  data  for  B-.  cloacae  and  for  B.  faecalis-alkaligenes  indicate  that 
these  two  organisms  are  comparable  with  respect  to  their  activity  in 
the  gelatin  medium.  The  amino-acid  concentrations  are  low  in  both 
cases.  B.  cloacae,  however,  forms  comparatively  large  amounts  of  free 
ammonia,  the  rate  of  formation  being  slightly  higher  in  the  sugar- 
free  culture.  The  amino-acid  figures  do  not  differ  greatly  in  the  two 
cultures  of  this  organism.  Considering  the  well-known  utilization  of 
glucose  by  B.  cloacae,  it  is  surprising  that  its  protein-sparing  action  is 
so  little  marked  in  this  case.  B.  faecalis-alkaligenes  lives  up  to  its  repu- 
tation in  these  cultures,  also,  in  showing  no  decrease  in  its  ammonia- 
production  in  the  presence  of  glucose. 

The  two  sets  of  cultures  of  B.  pyocyaneus,  the  one  grown  under 
aerobic,  the  other  under  anaerobic  conditions,  furnish  an  instructive 
comparative  study  in  the  biochemistry  of  micro-organisms.  The  sur- 
prising fact  in  these  data  is  that  so  little  difference  is  shown  between 
the  two  sets.  On  the  whole  the  greater  chemical  activity  seems  to  be 
shown  in  the  cultures  grown  without  the  exclusion  of  oxygen.  This 
difference  is  apparent,  however,  only  during  the  first  half  of  the  period 
of  the  experiment.  The  effect  of  glucose  is  greater  in  the  aerobic 
than  in  the  anaerobic  cultures.  As  multiplication  appeared  to  be  much 
slower  in  the  latter,  it  is  very  likely  that  the  differences  which  do 
exist  are  to  be  ascribed  to  differences  in  the  numbers  of  organisms 
rather  than  to  actual  differences  in  the  course  or  extent  of  chemical 
change  due  to  the  presence  or  absence  of  oxygen.  Likewise,  the 
apparently  lowered  protein-sparing  effect  of  glucose  in  the  anaerobic 
cultures  is  probably  due  to  a  difference  between  the  numbers  of  organ- 
isms, for  there  was  an  unquestionably  heavier  growth  in  the  anaerobic 
culture  containing  the  sugar  than  in  the  sugar-free  culture  grown 
under  the  same  conditions. 

The  cultures  of  B.  welchii  show  this  effect  of  glucose  very  plainly. 
The  enormously  greater  chemical  activity  of  this  organism  when  grown 
with  glucose  can  only  be  attributed  to  greatly  increased  multiplication 
in  the  presence  of  glucose.  The  appearance  of  the  cultures  fully  sub- 
stantiates this  conclusion. 

It  will  be  observed  from  the  data  that  the  proteolytic  activity  of  B. 
welchii  when  grown  under  favorable  conditions  is  considerable.  As 
will  be  pointed  out  later,  the  nitrogen  determined  in  the  form  of 
amino-acid  and  ammonia  cannot  be  assumed  to  represent  nearly  all  the 
nitrogen  which  is  removed  from  its  combinations  in  the  protein  mole- 
cule to  form  simpler  compounds.     When  we  consider,  therefore,  that 


28  H.     J.     Sears 

on  the  15th  day  of  incubation  the  nitrogen  so  determined,  amounts  to 
nearly  50%  of  the  total  nitrogen  of  the  medium,  we  can  scarcely  agree 
with  Rettger's*^  observation  that  this  bacillus  is  primarily  a  fermenting 
organism. 

The  initial  reduction  in  amount  of  the  free  ammonia  which  is 
originally  present  in  the  medium  occurs  in  all  of  the  cultures  except 
the  sugar-free  culture  of  B.  subtilis.  The  probable  reason  for  this 
exception  has  already  been  suggested.  In  all  cases  this  initial  reduction 
takes  place  until  the  ammonia  value  actually  reaches  zero,  and  there- 
after, in  a  number  of  the  cultures,  this  compound  remains  absent  for 
several  days.  This  peculiar  behavior  was  not  shown  by  the  pure  pep- 
tone cultures  and  therefore  it  must  be  ascribed  to  the  presence  of 
gelatin.  Obviously,  the  amino-acids  resulting  from  the  decomposition 
of  this  protein  are  broken  down  to  the  form  of  ammonia  to  a  much 
smaller  extent  than  those  resulting  from  the  splitting  of  Witte's  pep- 
tone. Probably  in  the  presence  of  an  abundance  of  nourishment  the 
decomposition  is  not  carried  so  far. 

EVIDENCE   OF   THE   EXISTENCnf  OF    CONSIDERABLE    QUANTITIES    OF 

NITROGEN   IN    COMPOUNDS   INTERMEDIARY   BETWEEN    AMINO- 

ACID    AND    AMMONIA 

Investigators  who  have  speculated  on  the  manner  in  which  the 
amino-acids  resulting  from  protein-decomposition  by  micro-organisms 
are  further  broken  down  in  the  course  of  putrefaction  have  taught  us 
to  suppose  that  the  first  reaction  to  take  place  is  deaminization  with 
the  splitting  off  of  ammonia.*^  This  would  mean,  of  course,  that  all 
the  nitrogen  of  that  group  determined  by  the  Van  Slyke  method,  i.  e., 
the  alpha  amino  nitrogen,  is  transformed  into  ammonia.  That  this  is 
not  true  in  the  case  of  the  cultures  investigated  by  us  becomes  evident 
when  the  tables  are  studied  more  carefully.  Every  decrease  in  amino- 
acid  content  which  occurs  in  the  interval  between  two  analyses  must 
represent,  of  course,  the  minimal  amount  of  this  form  of  nitrogen 
which  has  been  altered  during  the  period.  If  such  alteration  con- 
sisted in  large  part  or  entirely  in  deaminization,  then  the  decrease 
should  appear  as  a  corresponding  increase  in  ammonia  nitrogen.  The 
instances  in  which  this  is  true  in  the  data  given  are  so  rare  as  to  be 
considered  purely  accidental.  That  this  extra  amount  of  amino-acid 
nitrogen  metabolized  could  be  to  any  considerable  degree  accounted 
for  by  ammonia  lost  through  volatilization  is  made  improbable  from 
the  following  experiment:  Five  cubic  centimeters  of  a  2%  peptone 

«»  Jour.  Biol.  Chem.,  1908,  4,  p.  45. 

"  Lafar:   Handbuch  der  technischen   Mykologie,   1904-1906,   3,  p.    103. 


Nitrogen     Metabolism     of    Bacteria  29 

solution  were  placed  in  each  of  6  tubes,  sterilized,  and  inoculated  sev- 
erally with  the  organisms  as  indicated  in  Table  17.  After  15  days' 
incubation  at  Z7  C.  the  tubes  were  again  sterilized  and  the  whole 
contents  of  each  submitted  to  a  total  nitrogen-determination.  The 
results  are  given  in  Table  17.  The  figures  represent  the  total  nitrogen 
in  milligrams  in  the  5  c.c.  of  the  corresponding  culture. 

TABLE     17 
Nitrogen   Lost   from   Bacterial  Cultures  by  Volatilization 


Organism 


Milligrams  of  Total 

Nitrogen  in  5  c.c.  of 

Culture  Medium  After 

15  Days'  Incubation 


Sterile  control 

B.  coli-communis 

B.  typhosus 

B.  pyoeyaneus 

B.  faecalis-alkaligenes. 
B.  subtilis 


15.95 
15.64 
15.94 
15.61 
15.72 
14.39 


It  is  seen  that  B.  subtilis  is  the  only  one  of  the  organisms  investi- 
gated in  this  respect  which  lost  any  appreciable  quantity  of  nitrogen 
during  the  15  days'  incubation.  Even  its  loss  could  by  no  means 
account  for  the  differences  mentioned.  Berghaus*^  also  gives  data  on 
the  loss  of  ammonia  through  volatilization.  Tho  ammonia  is  undoubt- 
edly utilized  to  a  certain,  extent  by  most  of  the  species  studied,  the 
amount  utilized  must  necessarily  be  small.  That  part  of  it  which 
is  used  for  the  synthesis  of  bacterial  protein  or  the  metabolism  of 
which  results  in  compounds  which  are  not  volatile  could  not  be  great 
enough  to  be  considered  in  the  present  connection. 

The  only  conclusion  which  can  be  drawn  from  the  facts  in  the 
case,  therefore,  is  that  the  greater  part  of  the  reported  losses  in  amino- 
acid  nitrogen  which  take  place  from  time  to  time  is  due  to  the  con- 
version of  this  nitrogen  into  compounds  other  than  ammonia.  Further, 
the  continued  low  concentration  or  the  entire  absence  of  the  latter,  as 
seen  particularly  in  the  gelatin  cultures,  during  the  first  days  of  the 
experiments,  points  to  the  conclusion  that  ammonia  arises  from  the 
decomposition  of  these  other  compounds,  and  that  the  latter  are  there- 
fore to  be  regarded  as  important  stages  in  the  complete  decomposition 
of  protein.  That  some  of  them  are  true  end  products,  and  undergo 
no  further  change  through  the  action  of  the  micro-organism  concerned 
is  evidenced  by  the  slight  extent  to  which  creatin  is  attacked  by  B. 
proteus  (Table  5),  which,  as  seen  in  Table  1,  is  capable  of  forming 
this  substance  from  peptone. 

*-  Arch.  f.  Hyg.,  1907,  64,  p.   1. 


30 


H.     J.     Sears 


THE   FORMATION   BY   BACTERIA   OF   UREA,    URIC   ACID,   ALLANTOIN, 
CREATIN,  AND  CREATININ 

Urea  and  Uric  Acid. — All  the  peptone  cultures  described  thus  far 
in  this  paper  were  examined  in  several  stages  of  their  growth  for  urea 
and  uric  acid.    Neither  of  these  compounds  was  found  in  any  case. 

The  urease  method  originated  by  Van  Slyke"  was  used  for  the  detection  of 
the  former,  and  the  colorimetric  method  used  by  Folin  and  Denis,"  for  the 
latter.  Both  these  methods  were  proved  to  be  efficient  by  determinations  made 
on  cuhures  to  which  weighed  amounts  of  the  pure  chemicals  had  been  added. 
The  medium  used  in  the  case  of  uric  acid  was  a  solution  containing  10  gm. 
asparagin,  2.5  gm.  NaiCOa,  2  gm.  Na2HP04,  traces  of  MgSO*  and  CaCU  and 
0.902  gm.  of  uric  acid  per  liter.  The  time  of  incubation  was  15  days.  The 
medium  used  in  the  case  of  urea  was  a  2%  peptone  solution  containing  about 
10  gm.  of  urea  per  liter.     The  cultures  were  incubated  for  7  days. 

TABLE     18 
Decomposition  of  Uric  Acid  by  Bacteria 


Organism 


Sterile  control.... 
B.  coli-communis 

B.  acidi-laetici 

B.  pyocyaneus.. . 

B.  smegmae 

Sp.  choleras 


Ammonia  Nitro- 
gen per  100  c.c. 
Culture  Medium 


5.3  mgm. 
112.0  mgm. 
102.6  mgm. 

39.1  mgm. 

43.2  mgm. 
41.5  mgm. 


Amino-acid  Nitro- 
gen per  100  c.c. 
Culture  Medium 


94.5  mgm. 

36.0  mgm. 

7.3  mgm. 

22.5  mgm. 
68.0  mgm. 

32.6  mgm. 


Uric  Acid 

per  100  c.c. 

Culture  Medium 


65.7  mgm. 
7.5  mgm. 
None 
None 
None 
None 


TABLE     19 
Decomposition  of  Urea  by  Bacteria* 


Organism 

Ammonia 

Nitrogen 

in  5  c.c. 

Culture  Fluid 

Urea 

Nitrogen 

In  5  c.c. 

Culture  Fluid 

Nitrogen  of  Urea 
Decomposed 

in  5  c.c. 
Culture  Fluid 

None 
3.03  mgm. 
3.12  mgm. 
2.31  mgm. 
0.52  mgm. 
2.24  mgm. 

22.28  mgm. 
21.75  mgm. 
21.23  mgm. 
15.48  mgm. 
6.73  mgm. 

None 

0.53  mgm. 

1.05  mgm. 

B.  acidi-lactici 

6.S0  mgm. 

15.45  mem. 

6.37  mgm.                15.81   mem. 

*  In  this  test  inoculations  were  made  into  5-c.c.  portions  of  the  culture  medium,  and  the 
tubes  were  incubated  tor  7  days. 

It  is  not  surprising  that  uric  acid  and  urea  are  not  to  be  found  in 
bacterial  cultures  when  it  is  considered  how  easily  and  completely  both 
are  decomposed  by  bacteria.  Tables  18  and  19  give  the  results  of  brief 
tests  designed  to  show  this. 

Allantoin. — Numerous  attempts  were  made  to  prove  the  presence 
of  allantoin  in  cultures  on  pure  peptone  solutions  as  well  as  in  those 
on  peptone  solutions  containing  uric  acid,  but  it  was  impossible  to  find 

"  Van  Slyke  and  Cullen:  Proc.  Soc.  Exper.  Biol,  and  Med.,  1913,  11,  p.  56. 
"  Jour.   Biol.  Chem.,  1913,  14,  p.  95. 


Nitrogen     Metabolism     of    Bacteria  31 

a  method  by  which  all  interfering  substances  were  excluded.  The 
Wiechowski"  method,  for  which  great  accuracy  is  claimed  in  the  deter- 
mination of  allantoin  in  urine,  was  employed  repeatedly,  but  without 
success. 

This  method  consists  in  precipitation  by  phosphotungstic  acid,  the  removal  of 
the  excess  of  this  reagent  by  means  of  lead  oxid  and  acetic  acid,  the  elimination 
of  the  chlorid  with  silver  acetate,  and  the  final  precipitation  of  all  heavy  metals 
with  hydrogen  sulfid.  After  removal  of  the  latter  by  aeration  the  solution  is 
made  alkaline  with  magnesium  oxid  and  the  allantoin  precipitated  by  means  of 
a  solution  consisting  of  20%  sodium  acetate  and  1%  mercuric  acetate. 

It  was  found  that  in  all  bacterial  cultures  on  peptone  media  a  precipitate  was 
obtained  at  this  point  which  had  a  varying,  in  some  cases  high,  total-nitrogen 
content.  Moreover,  the  sterile  control  usually  gave  a  precipitate  here  also, 
which  varied  from  a  slight  opalescence  to  an  appreciable  deposit.  It  was  not 
possible  to  prepare  the  characteristic  crystals  of  allantoin  from  any  of  these 
precipitates.  It  is  probable  that  the  latter  consisted,  in  the  most  part,  of  amino- 
acids  which  escaped  precipitation  by  the  phosphotungstic  acid.  Levene  and 
Beatty"  showed  that  many  of  these  acids  are  precipitated  completely  by  this 
reagent  only  when  it  is  present  in  very  high  concentration.  Such  concentrations 
would  be  entirely  impractical  in  the  present  connection. 

Attempts  to  show  that  uric  acid  is  decomposed  by  bacteria  with  the  forma- 
tion of  allantoin  were  unsuccessful  for  the  same  reasons  as  have  just  been 
outlined.  A  medium  having  asparagin  as  the  nitrogenous  base  was  also  used 
in  these  experiments.  The  Wiechowski  method  was  inapplicable  here  also,  since 
asparagin,  not  being  precipitated  by  phosphotungstic  acid,  is  brought  down  with 
the  allantoin  by  the  sodium-acetate  mercuric-acetate  reagent.  If  allantoin  was 
ever  present  in  this  precipitate,  it  was  there  in  such  small  quantities  that  it  could 
not  be  detected  in  the  presence  of  asparagin. 

This  inability  to  separate  allantoin  from  asparagin  would  seem  to  render 
the  Wiechowski  method  inapplicable  also  to  the  determination  of  allantoin  in 
plants.  In  fact,  it  has  been  shown  recently  in  this  laboratory  that  a  very  large 
portion  of  the  nitrogen  determined  as  allantoin  in  plant  tissue  by  this  method 
is  in  reality  asparagin,  where  this  substance  is  present  in  considerable  amounts.* 

The  problem  of  the  formation  of  allantoin  by  bacteria  seems  to 
depend  for  its  solution  on  the  perfection  of  the  methods  for  the  deter- 
mination of  this  compound  and  its  separation  from  interfering  sub- 
stances. 

Creatin  and  Creatinin. — Most  of  the  peptone  cultures  or  peptone 
solutions  reported  in  the  first  part  of  this  paper  were  tested  for  creatin 
and  creatinin.  In  general,  the  results  were  in  close  agreement  with 
those  of  Fitzgerald  and  Schmidt. ^^  That  is,  only  B.  proteus  was  found 
to  form  appreciable  amounts  of  creatinin  on  solutions  of  peptone  alone. 
It  was  found  however  that  in  those  cultures  containing  glucose,  a  test 
for  this  substance  and  for  creatin  gave  positive  results  in  a  large  num- 

"  Neubauer's  Analyse  des  Harns,   1913,  2,  p.  1076. 

"«  Levene  and  Beatty,  Ztschr.   f.   physiol. '  Chem.,   1906,  47,  p.   149. 

*  Yet  unpublished  thesis  of  G.  C.   Swan,  Stanford  University,  May,  1915. 


32  H.     J.     Sears 

ber  of  cases.    More  careful  investigation  of  this  subject  was  considered 
important,  and  the  following  experiment  was  therefore  carried  out : 

A  large  amount  of  2%  peptone  solution  containing  0.5%  NaCl  was  divided  in 
half  and  to  one  portion  1%  glucose  was  added.  The  two  portions  were  placed 
in  sterile  flasks,  100  c.c.  to  each,  and  sterilized  in  the  autoclave  for  10  minutes. 
The  flasks  were  then  inoculated  and  placed  in  the  incubator  at  Zl  C.  Each  organ- 
ism to  be  tested  was  inoculated  into  a  flask  containing  glucose  and  into  one  not 
containing  glucose. 

Folin"  claimed  accuracy  for  his  methods  for  the  determination  of  creatin 
and  creatinin  in  the  presence  of  considerable  quantities  of  glucose.  Neverthe- 
less, it  was  considered  essential  to  maintain  two  sterile  controls,  one  with,  and 
one  without,  glucose.  Samples  were  withdrawn  from  each  of  the  cultures  and 
from  the  controls  at  definite  intervals  and  after  sterilization  in  steam  at  100  C, 
they  were  subjected  to  analysis  for  creatinin  and  creatin,  by  Folin's  methods. 

Tables  20  and  21  give  the  results  of  these  analyses.  The  values 
are  in  milligrams  per  100  c.c.  The  fact  that  neither  of  the  sterile  con- 
trols gave  at  any  time  a  color  reaction  seems  sufficient  to  establish  at 
once  the  noninterference  of  glucose  with  the  determinations.  We  may 
assume,  therefore,  that  the  figures  given  indicate  with  reasonable 
accuracy  the  comparative  creatinin-  and  creatin-forming  power  of  the 
micro-organisms  in  question. 

It  will  be  observed  that  only  B.  proteus  and  Sp.  cholerae  form  both 
creatin  and  creatinin  in  the  absence  of  sugar.  B.  subtilis  forms  creatin 
but  not  creatinin.  Of  the  cultures  on  the  glucose-containing  solutions, 
only  B.  faecalis-alkaligenes  fails  to  give  the  reaction  for  creatinin.  This 
culture  shows  on  the  second  day  a  considerable  quantity  of  creatin. 
Tests  for  this  substance  thereafter,  however,  are  all  negative. 

Examination  of  the  tables  will  suggest  that  there  are  several  possi- 
bilities for  the  diflferentiation  of  species  by  this  method.  For  exam- 
ple, on  the  second  day  the  amount  of  creatinin  in  the  culture  of  B. 
typhosus  is  5.9  mgm.  per  100  c.c,  an  amount  which  is  easily  detectable 
in  a  5-c.c.  portion.  The  corresponding  cultures  of  B.  coli  and  B. 
faecalis-alkaligenes  both  are  negative  in  the  test  for  creatinin.  Like- 
wise, Sp.  cholerae  gives  6.6  mgm.  of  creatinin  on  the  second  day,  while 
the  morphologically  and  culturally  similar  organism,  Sp.  metchnikovii, 
gives  only  a  trace.  It  is  interesting  to  note  that  in  the  point  of  their 
creatin-production  all  five  of  the  organisms  mentioned,  show  just  the 
reverse  characteristics;  the  cultures  of  B.  coli,  B.  faecalis-alkaligenes, 
and  Sp.  metchnikovii  show  high  concentration,  while  those  of  B. 
typhosus  and  Sp.  cholerae  give  low  values  for  this  compound. 

In  most  of  the  cultures  the  maximal  concentration  of  both  creatin 
and  creatinin  seemed  to  be  reached  from  about  the  5th  to  the  8th  days. 

*'  Jour.   Biol.  Chem.,  1914,  17,  p.  475. 


Nitrogen     Metabolism     of     Bacteria 


33 


After  that  the  amount  seemed  to  suffer  some  decrease.  In  the  case 
of  B.  pyocyaneus  and  B.  subtilis  a  maximum  is  not  shown  in  the  time 
of  the  experiment.  The  creatinin  content  in  the  culture  of  Staph, 
pyogenes  shows  a  continuous  increase,  but  its  creatin  content  falls 
nearly  to  zero  after  the  second  day. 

TABLE     20 
Formation  of  Creatinin  in  Peptone  Cultures  of  Micro-organisms 


Culture 


2  Days  Old 

5  Days  Old 

!• 

2* 

1 

2 

_ 

3.4 

Trace 

4.5 

— 

4.0 

— 

6.3 

— 

5.9 

— 

8.0 

— 

— 

— 

5.2 

— 

6.6 

— 

8.8 

— 

— 

— 

5.6 

— 

5.1 

— 

7.3 

— 

3.9 

— 

4.4 



Trace 



Trace 

— 

Trace 

— 

3.5 

— 

8.8 

— 

4.1 

— 

3.7 

— 

4.2 

— 

Trace 

— 

3.2 

~ 

~ 

~ 

~ 

B.  proteus-vulgaris 

B.  pyocyaneus 

B.  typhosus 

B.  coll-communis 

Sp.  cholerae 

B.  subtilis 

B.  paratyphosus 

B.  cloacae 

B.  faecalis-alkaligenes. 

Sp.  metchnikovii 

Staph,  pyogenes 

B.  dysenteriae,  Shiga.. 

B.  acidi-lactici 

B.  prodigiosus 

Sterile  controls 


*  The  numbers   1    and   2   refer  to   the  glucose-free   and   the   glucose-containing   cultures, 
respectively. 

TABLE     21 
Formation   of   Creatin   in   Peptone   Cultures   of   Micro-organisms 


Culture 


B.  proteus-vulgaris 

B.  pyocyaneus 

B.  typhosus 

B.  coli-communis 

Sp.  cholerae 

B.  subtilis 

B.  paratyphosus 

B.  cloacae 

B.  faecalis-alkaligenes. 

Sp.  metchnikovii 

Staph,  pyogenes 

B.  dysenteriae,  Shiga.. 

B.  acidi-lactici 

B.  prodigiosus 

Sterile  controls 


2  Days  Old 

5  Days  Old 

!• 

2» 

1 

2 



2.2 

Trace 

3.9 

— 

6.8 

— 

15.7 

— 

3.0 

— 

6.4 

— 

7.2 

■  — 

4.8 

— 

2.3 

— 

8.6 

— 

6.7 

— 

7.7 



2.4 

— 

5.3 

— 

8.9 

— 

6.0 

— 

9.8 

— 



— 

10.1 



4.3 

— 

7.8 



1.5 



7.3 

— 

1.9 



5.4 



2.8 

— 

10.7 

— 

2,1 

~ 

~ 

~ 

~ 

8  Days  Old 


5.9 


4.7 
72 


5.3 
t 
5.5 
5.3 
6.5 

*«!6" 
8.8 

0.4 

0.8 


14  Days  Old 


8.5 

3.1 

t 

t 

8.9 



t 

t 

18.1 

z 

4.{i 

— 

7.5 



1.8 



5.8 

— . 

8.2 

— 

7.1 

*  The  numbers  1  and  2  refer  to  the  glucose-free  and  the  glucose-containiDg  cultures, 
respectively. 

f  When  the  reagent  was  added  to  these  samples,  a  very  dark  color  was  produced,  which 
could  not  be  matched  with  the  standard  creatinin  solution. 

There  seem  to  be  two  possible  explanations  of  this  surprising  effect 
of  glucose  on  creatin-  and  creatinin-production  by  micro-organisms. 
Either  these  substances  are  regularly  produced  by  bacteria  in  peptone 
solutions  and  their  decomposition  as  fast  as  formed  prevented  by  the 
presence  of  sugar,  or  else  glucose,  o;-  some  of  its  split  products,  actually 
takes  part  in  the  synthesis  by  the  organisms  of  these  two  nitrogenous 


34 


H.    J.     Seaks 


compounds.  The  former  seems  to  be  the  more  probable  of  the  two 
hypotheses.  Fortunately,  this  proposition  is  susceptible  of  proof;  at 
least,  strong  evidence  for  or  against  it  may  be  obtained  by  investigating 
the  effect  of  glucose  on  the  decomposition  by  micro-organisms  of  quan- 
tities of  creatin  and  creatinin  added  to  peptone  solution. 

TABLE     22 

The  Decomposition  of  Creatinin  by  Bacteria 


Culture 


2  Days  Old 


Glucose  I  Glucose 
Present  |  Absent 


6  Days  Old 


Glucose  I  Glucose 
Present  I  Absent 


15  Days  Old 


Glucose    Glucose 
Present     Absent 


B.  proteus-vulgaris 

B.  pyocyaneus 

B.  typhosus 

B.  coli-communis 

B.  subtilis 

B.  faecalis-alkaligenes.. 
Staph,  pyogenes-aureus 
B.  dysenteriae,  Shiga... 

B.  acidi-lactici 

Sterile  medium 


52.4 
51.3 
48.1 
41.3 
41.8 
56.5 
47.4 
56.9 
50.7 
54.6 


47.1 
53.3 
53.9 
56.2 
64.2 
54.1 
13.4 
53.6 
45.2 
54.6 


29.3 
42.0 
33.6 
51.8 
46.8 
48.7 
48.1 
53.2 
55.6  - 


31.9 
38.3 
42.4 
44.6 
53.6 
55.5 
Trace 
51.4 
32.7 
55.5 


82.3 

* 

48.2 
32.9 
54.8 
48.8 
44.1 
51.8 
58.2 
55.1 


32.3 
Trace 
50.0 
Trace 
57.1 
52.2 
Trace 
51.6 
34.8 
55.3 


*  The  solution  was  too  dark-colored  to  be  comparable  with  the  standard. 

An  experiment  similar  to  the  one  just  described,  but  with  a  2%  peptone  solu- 
tion containing  in  each  liter  the  juice  from  1  lb.  of  lean  beef  as  the  medium 
used,  was  carrried  out.  Only  creatinin  determinations  were  made.  The  same 
method  was  used  as  in  the  former  experiment.  Samples  of  2  c.c.  were  used  for 
most  of  the  determinations. 

Table  22  gives  the  results  of  the  test.  As  in  all  the  preceding  tables, 
the  values  are  given  in  milligrams  per  100  c.c.  of  the  culture  fluid. 
The  data  seem  to  be  in  accord  with  the  explanation  already  suggested 
of  the  effect  of  glucose  on  the  formation  of  creatin  and  creatinin  by 
bacteria.  The  latter  compound  is  decomposed  in  most  cases  to  a  con- 
siderably less  extent  when  glucose  is  present  in  the  medium.  The  very 
great  eft'ect  of  this  substance  in  the  case  of  the  staphylococcus  cultures 
is  worthy  of  special  mention.  It  is  also  interesting  that  the  cultures 
of  B.  faecalis-alkaligenes  and  of  B.  dysenteriae  show  the  same  absence 
of  the  sparing  effect  of  sugar  as  they  did  in  the  case  of  the  peptone 
solutions. 

Further  experiments  are  now  in  progress  on  this  phase  of  bac- 
terial metabolism,  and  it  is  to  be  hoped  that  more  definite  information 
may  be  obtained  on  the  part  played  by  glucose  in  these  reactions. 

SUMMARY 

Peptone  cultures  of  most  bacteria  give  fluctuating  concentrations 
of  amino-acid,  showing  that  these  bodies  are  formed  and  broken  down 
continuously  by  the  organisms. 


Nitrogen     Metabolism    of    Bacteria  35 

A  few  strongly  proteolytic  organisms  are  exceptions  to  the  rule  in 
that  their  cultures  show  steadily  increasing  concentrations  of  amino- 
acid.  Among  these  are  B.  pyocyaneus,  B.  subtilis,  Sp.  cholerae,  and 
Sp.  metchnikovii. 

Most  species,  when  grown  in  peptone  or  peptone  gelatin  media, 
show  an  inclination  to  utilize  the  simpler  compounds  of  nitrogen  before 
attacking  the  protein  or  peptone. 

Most  species  also  show  evidences  of  a  continuous  utilization  of 
ammonium  salts  in  small  amounts. 

The  general  phenomenon  of  the  protein-sparing  effect  of  glucose 
is  evident  in  most  cultures  not  only  from  their  concentrations  of  free- 
ammonia,  but  also  from  their  concentrations  of  amino-acid,  and,  in 
fact,  may  be  disclosed  by  the  latter  when  the  former  fails  to  give  evi- 
dence of  it,  as  is  true  in  the  case  of  B.  faecalis-alkaligenes  and  B.  dysen- 
teriae,  Shiga. 

Practically  the  same  characteristics  of  ammonia-  and  amino-acid- 
production  are  shown  by  the  organisms  on  peptone  solutions  containing 
5%  gelatin  as  on  pure  peptone  solutions,  except  that  the  concentra- 
tions in  the  former  media  are  much  greater  in  the  case  of  those  organ- 
isms having  a  gelatin-liquefying  power. 

The  ammonia  and  amino-acid  curves  of  B.  pyocyaneus  grown 
aerobically  do  not  differ  materially  from  those  of  the  same  organism 
grown  anaerobically. 

B.  welchii,  when  grown  under  favorable  conditions,  shows  very 
strong  proteolytic  activity. 

The  free  ammonia  and  amino-acid  curves  of  most  micro-organ- 
isms give  evidence  of  the  existence  in  their  cultures  of  large  amounts 
of  nitrogenous  products  intermediary  between  amino-acid  and 
ammonia. 

Urea  and  uric  acid  are  not  found  in  peptone  cultures  of  bacteria, 
probably  because  of  the  ease  with  which  these  substances  are  decom- 
posed by  most  species. 

No  method  could  be  found  for  the  detection  and  determination  of 
allantoin  which  was  applicable  to  peptone  cultures  or  to  cultures  con- 
taining asparagin. 

A  few  species  of  bacteria  are  capable  of  producing  creatin  and 
creatinin  in  sugar-free  peptone  cultures.  Many  more  are  capable  of 
producing  these  substances  in  peptone  media  containing  glucose,  the 
probable  reason  for  this  effect  of  the  sugar  being  its  sparing  action  for 
the  two  compounds  in  question. 


This  investigation  was  carried  on  in  the  chemical  and  bacteriological  labora- 
tories of  Leland  Stanford  University  under  the  direction  of  Prof.  R.  E.  Swain. 


365230 


UNIVERSITY  OF  CALIFORNIA  UBRARY 


